PMC 20230813 pmc.key 5063996 CC BY no 2 2 Mechanism of Arabinoxylanase 10.1074/jbc.M116.743948 5063996 27531750 M116.743948 22149 42 cellulosome crystallography enzyme kinetics enzyme mechanism glycoside hydrolase Author's Choice—Final version free via Creative Commons CC-BY license. 22159 surname:Labourel;given-names:Aurore surname:Crouch;given-names:Lucy I. surname:Najmudin;given-names:Shabir surname:Baslé;given-names:Arnaud surname:Cuskin;given-names:Fiona surname:Brás;given-names:Joana L. A. surname:Jackson;given-names:Adam surname:Rogowski;given-names:Artur surname:Gray;given-names:Joseph surname:Yadav;given-names:Madhav P. surname:Henrissat;given-names:Bernard surname:Fontes;given-names:Carlos M. G. A. surname:Gilbert;given-names:Harry J. TITLE front 291 2016 0 The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans* 0.99930334 protein_type cleaner0 2023-09-18T10:24:23Z MESH: Arabinoxylanases protein_state DUMMY: cleaner0 2023-09-18T10:47:12Z Highly Decorated 0.9991499 chemical cleaner0 2023-09-18T10:24:31Z CHEBI: Xylans ABSTRACT abstract 79 The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack. 0.9984566 taxonomy_domain cleaner0 2023-09-18T10:24:41Z DUMMY: plant 0.99672145 chemical cleaner0 2023-09-18T10:24:48Z CHEBI: Xylan 0.99865866 taxonomy_domain cleaner0 2023-09-18T10:24:41Z DUMMY: plant 0.9991608 chemical cleaner0 2023-09-18T10:24:55Z CHEBI: β-1,4-xylose 0.99928457 chemical cleaner0 2023-09-18T10:25:01Z CHEBI: Xylp 0.99911076 chemical cleaner0 2023-09-18T10:25:08Z CHEBI: arabinofuranose 0.99916506 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9825443 protein_type cleaner0 2023-09-18T10:25:23Z MESH: penta-modular enzyme 0.99902785 protein cleaner0 2023-09-18T10:25:29Z PR: CtXyl5A 0.9981812 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.9987552 evidence cleaner0 2023-09-18T10:25:48Z DUMMY: crystal structure 0.9991007 protein_type cleaner0 2023-09-18T10:25:56Z MESH: arabinoxylanase 0.9980863 protein_state cleaner0 2023-09-18T10:26:03Z DUMMY: in complex with 0.50939804 chemical cleaner0 2023-09-18T10:26:08Z CHEBI: ligands 0.9991667 structure_element cleaner0 2023-09-18T10:26:19Z SO: catalytic domain 0.99892145 structure_element cleaner0 2023-09-18T10:26:25Z SO: non-catalytic carbohydrate binding module 0.99877536 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: crystal structure 0.99855906 evidence cleaner0 2023-09-18T10:26:12Z DUMMY: structure 0.9979739 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.998847 chemical cleaner0 2023-09-18T10:27:35Z CHEBI: Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp 0.9983198 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9865536 chemical cleaner0 2023-09-18T10:26:32Z CHEBI: xylose 0.9988514 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9991247 site cleaner0 2023-09-18T13:55:26Z SO: pocket 0.9987526 site cleaner0 2023-09-18T13:55:19Z SO: −2* subsite 0.9985621 site cleaner0 2023-09-18T10:28:03Z SO: catalytic center 0.99883324 site cleaner0 2023-09-18T13:55:30Z SO: −2* subsite 0.9991928 chemical cleaner0 2023-09-18T10:25:01Z CHEBI: Xylp 0.9991721 chemical cleaner0 2023-09-18T13:38:24Z CHEBI: Arap 0.9986651 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9984883 chemical cleaner0 2023-09-18T10:26:41Z CHEBI: arabinose 0.99220085 experimental_method cleaner0 2023-09-18T10:26:49Z MESH: Alanine substitution 0.99950814 residue_name_number cleaner0 2023-09-18T10:26:53Z DUMMY: Glu68 0.99953973 residue_name_number cleaner0 2023-09-18T10:26:59Z DUMMY: Tyr92 0.99952006 residue_name_number cleaner0 2023-09-18T10:27:04Z DUMMY: Asn139 0.9983119 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9982514 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.99881476 site cleaner0 2023-09-18T13:55:33Z SO: −2* subsite 0.9989426 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9970607 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.99551225 protein_state cleaner0 2023-09-18T10:48:32Z DUMMY: solvent-exposed 0.9989027 protein cleaner0 2023-09-18T10:25:30Z PR: CtXyl5A 0.9959501 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9521629 protein_type cleaner0 2023-09-18T10:27:42Z MESH: endo-xylanase INTRO title_1 1757 Introduction INTRO paragraph 1770 The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential. 0.9988167 taxonomy_domain cleaner0 2023-09-18T10:24:41Z DUMMY: plant 0.9986657 taxonomy_domain cleaner0 2023-09-18T10:43:54Z DUMMY: microorganisms 0.9988594 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant INTRO paragraph 2425 An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars. 0.9986474 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.97633845 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.90193385 chemical cleaner0 2023-09-18T10:28:42Z CHEBI: polysaccharide 0.99882466 chemical cleaner0 2023-09-18T10:28:55Z CHEBI: β-1,4-d-xylose chemical CHEBI: cleaner0 2023-09-18T13:41:15Z pyranose 0.99762434 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.99699223 chemical cleaner0 2023-09-18T10:29:48Z CHEBI: 4-O-methyl-d-glucuronic acid 0.99859554 chemical cleaner0 2023-09-18T10:29:53Z CHEBI: GlcA 0.9988452 chemical cleaner0 2023-09-18T10:30:05Z CHEBI: α-l-arabinofuranose 0.99826837 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.92519265 chemical cleaner0 2023-09-18T10:28:43Z CHEBI: polysaccharide 0.99387217 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.99838805 chemical cleaner0 2023-09-18T13:38:30Z CHEBI: ferulic acid 0.72233874 chemical cleaner0 2023-09-18T10:29:25Z CHEBI: hemicellulose chemical CHEBI: cleaner0 2023-09-18T10:29:37Z lignin 0.48770082 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99851996 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.99830014 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.9969138 taxonomy_domain cleaner0 2023-09-18T10:43:46Z DUMMY: cereal 0.70049024 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99178773 chemical cleaner0 2023-09-18T13:38:37Z CHEBI: sugars 0.998679 chemical cleaner0 2023-09-18T10:29:15Z CHEBI: l- and d-galactose 0.9987858 chemical cleaner0 2023-09-18T10:29:11Z CHEBI: β- and α-Xylp 0.9947548 taxonomy_domain cleaner0 2023-09-18T10:43:46Z DUMMY: cereal 0.57896805 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99786454 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.93305 chemical cleaner0 2023-09-18T13:38:41Z CHEBI: sugars INTRO paragraph 3517 Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site. 0.99864966 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.99864155 taxonomy_domain cleaner0 2023-09-18T10:43:53Z DUMMY: microorganisms 0.65709233 protein_type cleaner0 2023-09-18T10:30:23Z MESH: polysaccharide-degrading enzymes 0.9984101 protein_type cleaner0 2023-09-18T10:30:27Z MESH: glycoside hydrolases 0.9986268 protein_type cleaner0 2023-09-18T10:30:38Z MESH: polysaccharide lyases 0.99855816 protein_type cleaner0 2023-09-18T10:30:43Z MESH: carbohydrate esterases 0.99858 protein_type cleaner0 2023-09-18T10:30:54Z MESH: lytic polysaccharide monooxygenases 0.99558616 protein_type cleaner0 2023-09-18T10:31:00Z MESH: carbohydrate active enzymes 0.99593437 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.99739957 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99709374 protein_type cleaner0 2023-09-18T10:31:07Z MESH: endo-acting xylanases 0.9923669 protein_type cleaner0 2023-09-18T10:31:19Z MESH: glycoside hydrolase 0.99262285 protein_type cleaner0 2023-09-18T10:31:27Z MESH: GH 0.9738185 protein_type cleaner0 2023-09-18T10:31:44Z MESH: 5 0.9936046 protein_type cleaner0 2023-09-18T10:31:49Z MESH: GH10 0.99484766 protein_type cleaner0 2023-09-18T10:31:54Z MESH: GH11 0.9940958 protein_type cleaner0 2023-09-18T10:31:59Z MESH: GH8 0.9956683 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.99571615 chemical cleaner0 2023-09-18T10:28:43Z CHEBI: polysaccharide 0.99883527 protein_type cleaner0 2023-09-18T10:32:36Z MESH: α-glucuronidases 0.9988317 protein_type cleaner0 2023-09-18T10:32:43Z MESH: α-arabinofuranosidases 0.9990785 protein_type cleaner0 2023-09-18T10:32:49Z MESH: esterases 0.99923325 protein_type cleaner0 2023-09-18T10:33:02Z MESH: xylanases 0.9843167 chemical cleaner0 2023-09-18T10:29:26Z CHEBI: hemicellulose 0.99851674 protein_type cleaner0 2023-09-18T10:33:15Z MESH: GH30 0.99904984 protein_type cleaner0 2023-09-18T10:33:19Z MESH: glucuronoxylanases 0.99920505 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.9013893 protein_state cleaner0 2023-09-18T10:33:40Z DUMMY: bound at 0.99698335 site cleaner0 2023-09-18T13:55:43Z SO: −2 0.94409853 chemical cleaner0 2023-09-18T10:29:53Z CHEBI: GlcA 0.9941329 protein_type cleaner0 2023-09-18T10:30:28Z MESH: glycoside hydrolases site SO: cleaner0 2023-09-18T13:56:02Z subsites −1 and +1 0.9901659 site cleaner0 2023-09-18T13:56:05Z SO: subsites 0.9971566 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.9978956 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.99816823 protein cleaner0 2023-09-18T10:25:30Z PR: CtXyl5A 0.9986105 species cleaner0 2023-09-18T10:33:35Z MESH: Clostridium thermocellum 0.99725866 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99785656 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.99804723 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.999025 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.9891261 protein_state cleaner0 2023-09-18T10:33:45Z DUMMY: bound in 0.99886334 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9914019 site cleaner0 2023-09-18T13:56:14Z SO: −1 subsite 0.99909043 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.99802303 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.99847084 site cleaner0 2023-09-18T13:56:18Z SO: subsites 0.99856865 site cleaner0 2023-09-18T10:27:57Z SO: active site INTRO paragraph 5280 CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases. 0.99918216 protein cleaner0 2023-09-18T10:25:30Z PR: CtXyl5A 0.67755586 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.9980315 structure_element cleaner0 2023-09-18T10:35:54Z SO: catalytic module 0.860335 structure_element cleaner0 2023-09-18T10:36:01Z SO: CtGH5 0.9982284 structure_element cleaner0 2023-09-18T10:36:07Z SO: non-catalytic carbohydrate binding modules 0.9514713 structure_element cleaner0 2023-09-18T10:36:11Z SO: CBMs 0.3509473 protein_type cleaner0 2023-09-18T10:36:28Z MESH: 6 0.6156626 structure_element cleaner0 2023-09-18T10:36:40Z SO: CtCBM6 0.29874778 protein_type cleaner0 2023-09-18T10:36:53Z MESH: 13 0.874645 structure_element cleaner0 2023-09-18T10:37:03Z SO: CtCBM13 0.6269602 protein_type cleaner0 2023-09-18T10:37:18Z MESH: 62 0.8562604 structure_element cleaner0 2023-09-18T10:37:35Z SO: CtCBM62 0.9989775 protein_type cleaner0 2023-09-18T10:37:53Z MESH: fibronectin type 3 0.9988217 structure_element cleaner0 2023-09-18T10:38:03Z SO: Fn3 0.99874604 structure_element cleaner0 2023-09-18T10:38:09Z SO: dockerin 0.999186 structure_element cleaner0 2023-09-18T10:38:04Z SO: Fn3 0.9520046 structure_element cleaner0 2023-09-18T13:51:22Z SO: ligand-binding modules 0.9833201 structure_element cleaner0 2023-09-18T10:38:20Z SO: cellulose-disrupting modules 0.9987042 structure_element cleaner0 2023-09-18T10:38:10Z SO: dockerin 0.99853694 complex_assembly cleaner0 2023-09-18T10:38:32Z GO: cellulosome 0.98729646 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.99846244 species cleaner0 2023-09-18T10:38:26Z MESH: C. thermocellum 0.57434464 structure_element cleaner0 2023-09-18T10:36:40Z SO: CtCBM6 0.6251633 structure_element cleaner0 2023-09-18T10:36:01Z SO: CtGH5 0.59856856 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.99911016 chemical cleaner0 2023-09-18T10:38:43Z CHEBI: d-galactopyranose 0.99909335 chemical cleaner0 2023-09-18T10:38:47Z CHEBI: l-arabinopyranose 0.98460996 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 structure_element SO: cleaner0 2023-09-18T10:38:04Z Fn3 0.9985752 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: crystal structure 0.99908304 protein_state cleaner0 2023-09-18T10:39:04Z DUMMY: mature 0.9991793 protein cleaner0 2023-09-18T10:25:30Z PR: CtXyl5A 0.99882346 protein_state cleaner0 2023-09-18T10:39:06Z DUMMY: lacking structure_element SO: cleaner0 2023-09-18T10:38:10Z dockerin 0.7470989 mutant cleaner0 2023-09-18T10:39:59Z MESH: CtXyl5A-Doc protein_state DUMMY: cleaner0 2023-09-18T10:26:04Z in complex with chemical CHEBI: cleaner0 2023-09-18T10:40:19Z ligands 0.9989073 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99873036 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.99821556 protein_type cleaner0 2023-09-18T10:31:54Z MESH: GH11 0.9986395 protein_type cleaner0 2023-09-18T10:35:35Z MESH: endo-xylanases zbc0441653440001.jpg F1 FIG fig_caption 6633 Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains. protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.7534902 structure_element cleaner0 2023-09-18T10:41:23Z SO: GH 0.9863507 structure_element cleaner0 2023-09-18T13:51:27Z SO: CBM 0.99045384 structure_element cleaner0 2023-09-18T13:51:31Z SO: CE 0.99766254 protein_type cleaner0 2023-09-18T10:31:20Z MESH: glycoside hydrolase 0.7450091 structure_element cleaner0 2023-09-18T10:42:37Z SO: carbohydrate binding module 0.9983574 protein_type cleaner0 2023-09-18T10:42:23Z MESH: carbohydrate esterase structure_element SO: cleaner0 2023-09-18T10:41:42Z Laminin_3_G 0.99794406 protein_type cleaner0 2023-09-18T10:41:46Z MESH: concanavalin A lectin superfamily 0.998723 structure_element cleaner0 2023-09-18T10:38:04Z SO: FN3 0.9979355 structure_element cleaner0 2023-09-18T13:51:37Z SO: fibronectin type 3 domain structure_element SO: cleaner0 2023-09-18T10:38:10Z dockerin RESULTS title_1 6993 Results RESULTS title_4 7001 Substrate Specificity of CtXyl5A 0.99469876 protein cleaner0 2023-09-18T10:25:30Z PR: CtXyl5A RESULTS paragraph 7034 Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration. 0.999027 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9967934 protein_type cleaner0 2023-09-18T13:34:39Z MESH: arabinoxylan-specific xylanase 0.9990753 chemical cleaner0 2023-09-18T10:45:39Z CHEBI: xylooligosaccharides 0.99847776 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.99852186 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.8807422 taxonomy_domain cleaner0 2023-09-18T10:44:28Z DUMMY: wheat 0.99000096 taxonomy_domain cleaner0 2023-09-18T10:44:41Z DUMMY: rye 0.99898416 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.99872786 chemical cleaner0 2023-09-18T10:45:23Z CHEBI: WAX 0.9978104 chemical cleaner0 2023-09-18T10:45:27Z CHEBI: RAX 0.9987662 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.99883765 site cleaner0 2023-09-18T13:56:24Z SO: pocket site SO: cleaner0 2023-09-18T13:56:40Z −2* 0.9989691 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.99868107 site cleaner0 2023-09-18T13:56:44Z SO: −1 subsite 0.99863005 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: Arabinose 0.99874973 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9984124 chemical cleaner0 2023-09-18T10:45:47Z CHEBI: oligosaccharides 0.99849904 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9846201 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.99893147 site cleaner0 2023-09-18T13:56:48Z SO: subsites 0.9989191 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.99832433 site cleaner0 2023-09-18T13:56:54Z SO: −2* pocket 0.99759704 protein_state cleaner0 2023-09-18T10:48:32Z DUMMY: solvent-exposed 0.9976566 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99891055 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9827283 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9425561 taxonomy_domain cleaner0 2023-09-18T10:43:47Z DUMMY: cereal 0.9973623 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5a 0.9663724 experimental_method cleaner0 2023-09-18T14:05:42Z MESH: incubated 0.9815502 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9964102 experimental_method cleaner0 2023-09-18T12:47:03Z MESH: TLC 0.9857853 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99912196 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9990427 chemical cleaner0 2023-09-18T10:29:53Z CHEBI: GlcA 0.99907273 chemical cleaner0 2023-09-18T10:46:20Z CHEBI: l-Gal 0.9989554 chemical cleaner0 2023-09-18T10:46:27Z CHEBI: d-Gal 0.999036 chemical cleaner0 2023-09-18T10:46:32Z CHEBI: d-Xyl 0.9979019 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9713136 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9989698 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A taxonomy_domain DUMMY: cleaner0 2023-09-18T10:45:08Z corn 0.9789818 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.9837578 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.99865055 protein_type cleaner0 2023-09-18T10:35:36Z MESH: endo-xylanases 0.99723804 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.9968777 protein_type cleaner0 2023-09-18T10:31:54Z MESH: GH11 0.7958819 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX protein_state DUMMY: cleaner0 2023-09-18T10:47:40Z lack of 0.99779606 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9989344 site cleaner0 2023-09-18T10:27:57Z SO: active site protein_state DUMMY: cleaner0 2023-09-18T13:02:58Z bind to 0.9935819 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.99850285 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.68566746 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.9984811 chemical cleaner0 2023-09-18T11:03:41Z CHEBI: oligosaccharides 0.99833727 site cleaner0 2023-09-18T13:56:58Z SO: subsites 0.9986953 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9970818 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9962427 protein_state cleaner0 2023-09-18T10:48:31Z DUMMY: solvent-exposed T1.xml T1 TABLE table_caption 8773 Kinetics of GH5_34 arabinoxylanases evidence DUMMY: cleaner0 2023-09-18T13:03:30Z Kinetics protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.99908423 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases T1.xml T1 TABLE table_caption 8809 ND, not determined; NA, no activity. T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="2" colspan="1">Enzyme</th><th align="center" rowspan="2" colspan="1">Variant</th><th align="center" rowspan="1" colspan="3"><italic>k</italic>_cat/<italic>K_m</italic><hr/></th></tr><tr><th align="center" rowspan="1" colspan="1">WAX</th><th align="center" rowspan="1" colspan="1">RAX</th><th align="center" rowspan="1" colspan="1">CX</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="3"><italic>min</italic>^{−<italic>1</italic>} <italic>mg</italic>^{−<italic>1</italic>} <italic>ml</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3-CBM62</td><td align="left" rowspan="1" colspan="1">800</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">460</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3</td><td align="left" rowspan="1" colspan="1">1,232</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">659</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13</td><td align="left" rowspan="1" colspan="1">1,307</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">620</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6</td><td align="left" rowspan="1" colspan="1">488</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">102</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: E68A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: Y92A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N135A</td><td align="left" rowspan="1" colspan="1">260</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N139A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ac</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">628</td><td align="left" rowspan="1" colspan="1">1,641</td><td align="left" rowspan="1" colspan="1">289</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Gp</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">2,600</td><td align="left" rowspan="1" colspan="1">9,986</td><td align="left" rowspan="1" colspan="1">314</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">D45A</td><td align="left" rowspan="1" colspan="1">102</td><td align="left" rowspan="1" colspan="1">203</td><td align="left" rowspan="1" colspan="1">23</td></tr></tbody></table> 8846 Enzyme Variant kcat/Km WAX RAX CX min−1mg−1ml CtXyl5A CtGH5-CBM6-CBM13-Fn3-CBM62 800 ND 460 CtXyl5A CtGH5-CBM6-CBM13-Fn3 1,232 ND 659 CtXyl5A CtGH5-CBM6-CBM13 1,307 ND 620 CtXyl5A CtGH5-CBM6 488 ND 102 CtXyl5A CtGH5-CBM6: E68A NA NA NA CtXyl5A CtGH5-CBM6: Y92A NA NA NA CtXyl5A CtGH5-CBM6: N135A 260 ND ND CtXyl5A CtGH5-CBM6: N139A NA NA NA AcGH5 Wild type 628 1,641 289 GpGH5 Wild type 2,600 9,986 314 VbGH5 Wild type ND ND ND VbGH5 D45A 102 203 23 0.98840487 evidence cleaner0 2023-09-18T13:50:05Z DUMMY: kcat 0.8297304 evidence cleaner0 2023-09-18T13:50:09Z DUMMY: Km chemical CHEBI: cleaner0 2023-09-18T10:45:23Z WAX chemical CHEBI: cleaner0 2023-09-18T10:45:28Z RAX chemical CHEBI: cleaner0 2023-09-18T10:46:39Z CX 0.96991134 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:27:01Z CtGH5-CBM6-CBM13-Fn3-CBM62 0.7872997 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:27:19Z CtGH5-CBM6-CBM13-Fn3 0.5415051 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:27:36Z CtGH5-CBM6-CBM13 0.60911536 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:27:53Z CtGH5-CBM6 0.47242165 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:28:09Z CtGH5-CBM6 0.5855907 mutant cleaner0 2023-09-18T10:49:08Z MESH: E68A 0.55555713 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:28:27Z CtGH5-CBM6 0.73202586 mutant cleaner0 2023-09-18T10:49:12Z MESH: Y92A 0.55746835 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:29:09Z CtGH5-CBM6 0.55628043 mutant cleaner0 2023-09-18T10:49:16Z MESH: N135A protein PR: cleaner0 2023-09-18T10:25:31Z CtXyl5A structure_element SO: cleaner0 2023-09-18T14:29:26Z CtGH5-CBM6 0.86166924 mutant cleaner0 2023-09-18T10:54:16Z MESH: N139A protein PR: cleaner0 2023-09-18T10:50:32Z AcGH5 0.9990313 protein_state cleaner0 2023-09-18T10:49:27Z DUMMY: Wild type 0.95552933 protein cleaner0 2023-09-18T12:47:40Z PR: GpGH5 0.99901223 protein_state cleaner0 2023-09-18T10:49:27Z DUMMY: Wild type protein PR: cleaner0 2023-09-18T10:49:41Z VbGH5 0.9988765 protein_state cleaner0 2023-09-18T10:49:26Z DUMMY: Wild type protein PR: cleaner0 2023-09-18T10:49:40Z VbGH5 0.9978617 mutant cleaner0 2023-09-18T10:49:22Z MESH: D45A RESULTS paragraph 9333 To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined. 0.76418567 protein_state cleaner0 2023-09-18T13:35:57Z DUMMY: bound only at 0.99534464 site cleaner0 2023-09-18T13:57:04Z SO: −2* 0.9800586 site cleaner0 2023-09-18T13:57:06Z SO: −1 0.9986288 site cleaner0 2023-09-18T13:57:09Z SO: negative subsites 0.9944119 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A chemical CHEBI: cleaner0 2023-09-18T10:46:39Z CX 0.93394166 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.99740463 experimental_method cleaner0 2023-09-18T10:59:02Z MESH: size exclusion chromatography 0.99753416 chemical cleaner0 2023-09-18T11:03:40Z CHEBI: oligosaccharides 0.9985832 experimental_method cleaner0 2023-09-18T10:58:49Z MESH: HPAEC 0.998097 chemical cleaner0 2023-09-18T13:38:46Z CHEBI: oligosaccharide chemical CHEBI: cleaner0 2023-09-18T13:39:31Z oligosaccharide chemical CHEBI: cleaner0 2023-09-18T13:39:32Z oligosaccharide 0.9654604 experimental_method cleaner0 2023-09-18T10:58:57Z MESH: Positive mode electrospray mass spectrometry chemical CHEBI: cleaner0 2023-09-18T12:16:00Z pentose chemical CHEBI: cleaner0 2023-09-18T12:16:00Z pentose 0.7560745 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide 0.8210523 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide 0.9970212 experimental_method cleaner0 2023-09-18T10:59:12Z MESH: TFA hydrolysis 0.9957487 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9935015 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9950472 chemical cleaner0 2023-09-18T11:03:41Z CHEBI: oligosaccharides 0.9897843 chemical cleaner0 2023-09-18T11:04:03Z CHEBI: disaccharides 0.9872656 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.97808754 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9635059 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose protein_type MESH: cleaner0 2023-09-18T12:15:03Z nonspecific arabinofuranosidase 0.9518543 protein cleaner0 2023-09-18T10:54:51Z PR: CjAbf51A chemical CHEBI: cleaner0 2023-09-18T13:39:31Z oligosaccharide 0.9971559 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9963881 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose chemical CHEBI: cleaner0 2023-09-18T11:03:20Z disaccharide 0.9972711 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9962746 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9963164 protein_type cleaner0 2023-09-18T10:55:06Z MESH: β-1,3-xylosidase 0.5966584 protein cleaner0 2023-09-18T10:55:11Z PR: XynB chemical CHEBI: cleaner0 2023-09-18T13:39:32Z oligosaccharide 0.9976882 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose chemical CHEBI: cleaner0 2023-09-18T11:03:20Z disaccharide 0.9992453 chemical cleaner0 2023-09-18T13:40:06Z CHEBI: β-1,3-xylobiose 0.97893924 protein_type cleaner0 2023-09-18T10:59:20Z MESH: β-1,4-specific xylosidase chemical CHEBI: cleaner0 2023-09-18T13:39:32Z oligosaccharide chemical CHEBI: cleaner0 2023-09-18T13:39:32Z oligosaccharide 0.9987957 site cleaner0 2023-09-18T13:57:13Z SO: −2* pocket 0.9992246 chemical cleaner0 2023-09-18T10:55:39Z CHEBI: β-1,4-xylooligosaccharides 0.999252 chemical cleaner0 2023-09-18T10:55:27Z CHEBI: Araf-Xylp 0.9992756 chemical cleaner0 2023-09-18T13:40:11Z CHEBI: Xyl-β-1,3-Xyl 0.9986477 site cleaner0 2023-09-18T13:57:16Z SO: −2 subsite 0.9981468 protein_type cleaner0 2023-09-18T10:31:07Z MESH: endo-acting xylanases 0.99076325 site cleaner0 2023-09-18T13:57:20Z SO: subsite 0.99634886 site cleaner0 2023-09-18T13:57:23Z SO: −2* 0.9985508 site cleaner0 2023-09-18T13:57:26Z SO: −2 subsite chemical CHEBI: cleaner0 2023-09-18T12:18:33Z (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl 0.9294588 chemical cleaner0 2023-09-18T11:03:41Z CHEBI: oligosaccharides chemical CHEBI: cleaner0 2023-09-18T10:25:42Z arabinoxylans zbc0441653440002.jpg F2 FIG fig_caption 11762 Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+. 0.99689424 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide 0.99468046 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.99636436 experimental_method cleaner0 2023-09-18T10:59:02Z MESH: size exclusion chromatography 0.9975695 experimental_method cleaner0 2023-09-18T10:58:50Z MESH: HPAEC 0.9924101 experimental_method cleaner0 2023-09-18T12:14:37Z MESH: ESI-MS protein_type MESH: cleaner0 2023-09-18T12:15:02Z nonspecific arabinofuranosidase 0.9880223 protein cleaner0 2023-09-18T10:54:52Z PR: CjAbf51A protein_type MESH: cleaner0 2023-09-18T12:15:31Z GH3 xylosidase 0.99361575 protein cleaner0 2023-09-18T10:55:12Z PR: XynB 0.99745184 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.99641883 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9981957 chemical cleaner0 2023-09-18T12:16:00Z CHEBI: pentose 0.76520646 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide 0.58194405 experimental_method cleaner0 2023-09-18T12:14:37Z MESH: ESI-MS 0.9983165 chemical cleaner0 2023-09-18T12:15:59Z CHEBI: pentose 0.96897036 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide RESULTS title_4 12357 Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands 0.9987371 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: Crystal Structure 0.998405 structure_element cleaner0 2023-09-18T10:35:55Z SO: Catalytic Module 0.95691925 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99861926 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in Complex with 0.46196067 chemical cleaner0 2023-09-18T13:40:17Z CHEBI: Ligands RESULTS paragraph 12434 To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars. 0.9303489 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9986395 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: crystal structure 0.9989794 site cleaner0 2023-09-18T12:17:04Z SO: substrate binding cleft 0.99862534 site cleaner0 2023-09-18T13:57:31Z SO: −2* subsite 0.99843544 evidence cleaner0 2023-09-18T13:50:13Z DUMMY: structure 0.7080453 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T14:29:50Z CtGH5-CtCBM6 0.9989565 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.99890447 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.99898124 protein_state cleaner0 2023-09-18T10:33:46Z DUMMY: bound in 0.99854136 site cleaner0 2023-09-18T13:57:34Z SO: −2* pocket 0.9988469 protein_state cleaner0 2023-09-18T12:17:09Z DUMMY: bound 0.9989888 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose chemical CHEBI: cleaner0 2023-09-18T13:41:14Z pyranose chemical CHEBI: cleaner0 2023-09-18T13:41:26Z furanose 0.9973653 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.99867976 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.998457 site cleaner0 2023-09-18T13:57:38Z SO: −1 subsite 0.9984829 protein_state cleaner0 2023-09-18T12:17:11Z DUMMY: bound 0.99893314 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9965403 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.9991899 chemical cleaner0 2023-09-18T13:40:21Z CHEBI: Arap 0.99857426 site cleaner0 2023-09-18T13:57:41Z SO: −2* subsite 0.99846435 site cleaner0 2023-09-18T13:57:44Z SO: pocket 0.99902916 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9983833 experimental_method cleaner0 2023-09-18T12:21:15Z MESH: soaking 0.99923503 protein_state cleaner0 2023-09-18T12:21:10Z DUMMY: apo 0.48547208 evidence cleaner0 2023-09-18T13:50:18Z DUMMY: crystals 0.99908113 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose chemical CHEBI: cleaner0 2023-09-18T13:40:56Z pentose 0.5487963 chemical cleaner0 2023-09-18T13:40:26Z CHEBI: sugar 0.9981328 protein_state cleaner0 2023-09-18T10:33:46Z DUMMY: bound in 0.9983646 site cleaner0 2023-09-18T13:57:47Z SO: −2* subsite chemical CHEBI: cleaner0 2023-09-18T13:41:15Z pyranose 0.9988084 evidence cleaner0 2023-09-18T13:50:23Z DUMMY: crystal structures 0.9991225 chemical cleaner0 2023-09-18T13:41:34Z CHEBI: β-1,3-xylobiose 0.9822428 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.9982703 chemical cleaner0 2023-09-18T11:03:20Z CHEBI: disaccharide site SO: cleaner0 2023-09-18T13:58:09Z −1 and −2* subsites 0.99872786 experimental_method cleaner0 2023-09-18T14:05:47Z MESH: co-crystallizing 0.99801016 protein_state cleaner0 2023-09-18T13:36:02Z DUMMY: nucleophile inactive 0.99902153 protein_state cleaner0 2023-09-18T13:36:07Z DUMMY: mutant 0.9535321 mutant cleaner0 2023-09-18T14:09:45Z MESH: CtGH5E279S structure_element SO: cleaner0 2023-09-18T10:36:40Z CtCBM6 0.98482054 chemical cleaner0 2023-09-18T10:45:23Z CHEBI: WAX 0.9992003 chemical cleaner0 2023-09-18T13:39:32Z CHEBI: oligosaccharide 0.9991468 chemical cleaner0 2023-09-18T13:41:37Z CHEBI: pentasaccharide 0.9987526 protein_state cleaner0 2023-09-18T12:19:16Z DUMMY: bound to 0.99912447 chemical cleaner0 2023-09-18T13:41:41Z CHEBI: β-1,4-xylotetraose 0.99879885 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9987872 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.99923956 chemical cleaner0 2023-09-18T13:41:45Z CHEBI: xylotetraose 0.9051479 site cleaner0 2023-09-18T13:58:13Z SO: subsites −1 to −4 0.9991596 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9983297 site cleaner0 2023-09-18T13:58:16Z SO: −2* pocket 0.9958412 evidence cleaner0 2023-09-18T13:50:27Z DUMMY: structures 0.99901795 chemical cleaner0 2023-09-18T13:40:22Z CHEBI: Arap 0.9989477 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9988612 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.9988979 protein_state cleaner0 2023-09-18T10:33:46Z DUMMY: bound in 0.99790555 site cleaner0 2023-09-18T13:58:18Z SO: −2* subsite 0.9976653 site cleaner0 2023-09-18T13:58:22Z SO: pocket 0.9838765 bond_interaction cleaner0 2023-09-18T12:20:15Z MESH: polar contact 0.9994537 residue_name_number cleaner0 2023-09-18T10:27:05Z DUMMY: Asn139 0.99667907 bond_interaction cleaner0 2023-09-18T12:20:11Z MESH: hydrogen bonding 0.999443 residue_name_number cleaner0 2023-09-18T10:27:05Z DUMMY: Asn139 0.99943477 residue_name_number cleaner0 2023-09-18T12:23:10Z DUMMY: Asn135 0.99941826 residue_name_number cleaner0 2023-09-18T14:06:25Z DUMMY: Gly136 0.99930656 residue_name_number cleaner0 2023-09-18T10:26:54Z DUMMY: Glu68 0.99909437 chemical cleaner0 2023-09-18T13:40:22Z CHEBI: Arap 0.9990483 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.9990252 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9971278 bond_interaction cleaner0 2023-09-18T12:19:57Z MESH: hydrogen bonds 0.99928504 residue_name_number cleaner0 2023-09-18T10:26:54Z DUMMY: Glu68 0.9993992 residue_name_number cleaner0 2023-09-18T10:26:59Z DUMMY: Tyr92 0.97813207 bond_interaction cleaner0 2023-09-18T12:20:03Z MESH: parallel interactions chemical CHEBI: cleaner0 2023-09-18T13:41:15Z pyranose chemical CHEBI: cleaner0 2023-09-18T13:41:27Z furanose zbc0441653440003.jpg F3 FIG fig_caption 14671 Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated. 0.99794537 site cleaner0 2023-09-18T13:58:28Z SO: −2* subsite 0.99496317 site cleaner0 2023-09-18T13:58:36Z SO: catalytic residues experimental_method MESH: cleaner0 2023-09-18T13:58:46Z mutated 0.9964224 residue_name cleaner0 2023-09-18T12:21:34Z SO: glutamate 0.9966878 residue_name cleaner0 2023-09-18T12:21:37Z SO: serine structure_element SO: cleaner0 2023-09-18T14:30:17Z CtGH5-CBM6 0.96348566 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.9985078 chemical cleaner0 2023-09-18T13:41:51Z CHEBI: arabinopyranose structure_element SO: cleaner0 2023-09-18T14:30:42Z CtGH5-CBM6 0.9178238 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.9992086 chemical cleaner0 2023-09-18T13:41:57Z CHEBI: xylopyranose 0.99710983 mutant cleaner0 2023-09-18T14:09:51Z MESH: CtGH5E279S structure_element SO: cleaner0 2023-09-18T10:51:34Z CBM6 0.9848954 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.99923825 chemical cleaner0 2023-09-18T13:42:01Z CHEBI: pentasaccharide 0.99916303 chemical cleaner0 2023-09-18T13:42:04Z CHEBI: β1,4-xylotetraose 0.999108 chemical cleaner0 2023-09-18T12:25:05Z CHEBI: l-Araf 0.9986161 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9975291 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.9933159 evidence cleaner0 2023-09-18T12:22:07Z DUMMY: Densities evidence DUMMY: cleaner0 2023-09-18T12:22:32Z maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ RESULTS paragraph 15430 The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase. 0.99893385 site cleaner0 2023-09-18T13:58:53Z SO: −2* pocket 0.9966867 experimental_method cleaner0 2023-09-18T12:23:04Z MESH: alanine substitution 0.9985077 protein_state cleaner0 2023-09-18T12:22:59Z DUMMY: mutants 0.9990114 mutant cleaner0 2023-09-18T10:49:08Z MESH: E68A 0.9989949 mutant cleaner0 2023-09-18T10:49:13Z MESH: Y92A 0.99898463 mutant cleaner0 2023-09-18T10:54:16Z MESH: N139A 0.99903464 protein_state cleaner0 2023-09-18T12:23:01Z DUMMY: inactive 0.99899477 site cleaner0 2023-09-18T13:58:56Z SO: −2* subsite 0.9990736 mutant cleaner0 2023-09-18T10:49:17Z MESH: N135A 0.99906486 protein_state cleaner0 2023-09-18T10:49:27Z DUMMY: wild type 0.9993063 residue_name_number cleaner0 2023-09-18T12:23:10Z DUMMY: Asn135 0.9992543 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.99923706 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9989507 site cleaner0 2023-09-18T13:58:59Z SO: −2* pocket protein_state DUMMY: cleaner0 2023-09-18T10:48:32Z solvent-exposed 0.99911714 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9991146 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.99835086 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9980102 chemical cleaner0 2023-09-18T10:26:33Z CHEBI: xylose 0.9981377 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose evidence DUMMY: cleaner0 2023-09-18T13:50:52Z kcat evidence DUMMY: cleaner0 2023-09-18T13:51:00Z Km 0.9909752 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9985014 chemical cleaner0 2023-09-18T10:45:23Z CHEBI: WAX 0.7723853 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.99858814 chemical cleaner0 2023-09-18T10:45:23Z CHEBI: WAX 0.99835324 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.926579 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.99903345 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase RESULTS paragraph 16441 In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously. 0.99896586 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.99930596 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9991194 chemical cleaner0 2023-09-18T12:23:44Z CHEBI: α-d-Xylp 0.99690115 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds 0.9994248 residue_name_number cleaner0 2023-09-18T12:23:59Z DUMMY: His253 0.9993506 residue_name_number cleaner0 2023-09-18T12:24:03Z DUMMY: Glu171 0.9857984 bond_interaction cleaner0 2023-09-18T12:20:16Z MESH: polar contact 0.99934465 residue_name_number cleaner0 2023-09-18T12:23:49Z DUMMY: Tyr255 0.9993443 residue_name_number cleaner0 2023-09-18T12:23:54Z DUMMY: Ser279 0.99672544 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds 0.9993864 residue_name_number cleaner0 2023-09-18T12:24:08Z DUMMY: Asn170 0.9994099 residue_name_number cleaner0 2023-09-18T10:26:59Z DUMMY: Tyr92 0.999243 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9981232 site cleaner0 2023-09-18T13:59:03Z SO: −2* subsite 0.9942347 bond_interaction cleaner0 2023-09-18T12:20:16Z MESH: polar contact 0.9994549 residue_name_number cleaner0 2023-09-18T10:27:05Z DUMMY: Asn139 0.9965925 bond_interaction cleaner0 2023-09-18T12:24:38Z MESH: hydrogens bonds 0.9993518 residue_name_number cleaner0 2023-09-18T12:23:50Z DUMMY: Tyr255 0.9992919 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.99828696 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.8674073 bond_interaction cleaner0 2023-09-18T12:24:31Z MESH: parallel apolar interactions 0.9994276 residue_name_number cleaner0 2023-09-18T12:24:22Z DUMMY: Phe310 0.9986855 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9959131 protein_state cleaner0 2023-09-18T13:36:24Z DUMMY: conserved 0.99930716 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99881834 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.9991805 protein_type cleaner0 2023-09-18T13:34:45Z MESH: endoglucanase 0.9994155 protein cleaner0 2023-09-18T13:45:00Z PR: BaCel5A zbc0441653440004.jpg F4 FIG fig_caption 17270 Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10. A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds. 0.99856913 site cleaner0 2023-09-18T13:59:11Z SO: negative subsites 0.9817981 mutant cleaner0 2023-09-18T14:10:02Z MESH: CtGH5E279S 0.8062502 structure_element cleaner0 2023-09-18T10:51:34Z SO: CBM6 0.9989459 protein_type cleaner0 2023-09-18T12:26:17Z MESH: cellulase 0.999137 protein cleaner0 2023-09-18T13:45:06Z PR: BaCel5A 0.99901605 protein_type cleaner0 2023-09-18T13:34:52Z MESH: xylanase 0.99872714 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.97036284 mutant cleaner0 2023-09-18T14:10:05Z MESH: CtGH5E279S 0.79618585 structure_element cleaner0 2023-09-18T10:51:34Z SO: CBM6 0.9977508 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.9992555 chemical cleaner0 2023-09-18T13:42:12Z CHEBI: pentasaccharide 0.99919766 chemical cleaner0 2023-09-18T13:42:14Z CHEBI: β1,4-xylotetraose 0.99904007 chemical cleaner0 2023-09-18T12:25:05Z CHEBI: l-Araf 0.9987269 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.9961971 bond_interaction cleaner0 2023-09-18T12:20:12Z MESH: hydrogen bonding 0.99668086 bond_interaction cleaner0 2023-09-18T12:25:53Z MESH: hydrophobic interactions 0.9985068 site cleaner0 2023-09-18T13:59:14Z SO: negative subsites 0.9700168 evidence cleaner0 2023-09-18T12:25:48Z DUMMY: density evidence DUMMY: cleaner0 2023-09-18T12:25:42Z maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ 0.999212 protein cleaner0 2023-09-18T13:45:09Z PR: BaCel5A 0.99365395 protein_state cleaner0 2023-09-18T10:26:04Z DUMMY: in complex with 0.9992079 chemical cleaner0 2023-09-18T13:42:17Z CHEBI: deoxy-2-fluoro-β-d-cellotrioside 0.9983261 protein cleaner0 2023-09-18T13:45:13Z PR: CmXyn10B protein_state DUMMY: cleaner0 2023-09-18T10:26:04Z in complex with 0.9992083 chemical cleaner0 2023-09-18T13:42:26Z CHEBI: xylotriose 0.9557879 mutant cleaner0 2023-09-18T14:10:11Z MESH: CtGH5E279S 0.9193694 structure_element cleaner0 2023-09-18T10:51:34Z SO: CBM6 0.99914455 protein cleaner0 2023-09-18T13:45:17Z PR: BaCel5A 0.9989845 protein_type cleaner0 2023-09-18T13:34:57Z MESH: xylanase 0.9984413 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.9951364 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds RESULTS paragraph 18283 The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G). 0.99838185 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99818134 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.7490116 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.9990195 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp 0.9982736 protein_state cleaner0 2023-09-18T10:48:32Z DUMMY: solvent-exposed 0.99916756 chemical cleaner0 2023-09-18T13:42:30Z CHEBI: xylotetraose 0.9988993 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.998987 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose site SO: cleaner0 2023-09-18T13:59:33Z subsites −2 to −4 0.99836254 protein_state cleaner0 2023-09-18T10:48:32Z DUMMY: solvent-exposed 0.9971443 chemical cleaner0 2023-09-18T13:41:15Z CHEBI: pyranose 0.846215 chemical cleaner0 2023-09-18T13:42:39Z CHEBI: sugars bond_interaction MESH: cleaner0 2023-09-18T14:07:27Z apolar interactions 0.9933171 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.98697215 site cleaner0 2023-09-18T13:59:38Z SO: −2 0.9984598 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp bond_interaction MESH: cleaner0 2023-09-18T14:07:11Z planar apolar interactions 0.99914765 chemical cleaner0 2023-09-18T10:25:15Z CHEBI: Araf 0.9965229 protein_state cleaner0 2023-09-18T12:19:16Z DUMMY: bound to 0.99492747 site cleaner0 2023-09-18T13:59:41Z SO: −2* subsite 0.9988463 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp site SO: cleaner0 2023-09-18T14:00:00Z subsites −2 and −3 0.9847268 bond_interaction cleaner0 2023-09-18T12:26:03Z MESH: hydrophobic contact 0.9995246 residue_name_number cleaner0 2023-09-18T14:06:30Z DUMMY: Val318 0.8751849 site cleaner0 2023-09-18T14:00:03Z SO: −3 0.9987112 chemical cleaner0 2023-09-18T10:25:02Z CHEBI: Xylp bond_interaction MESH: cleaner0 2023-09-18T14:00:26Z planar apolar interactions 0.99950683 residue_name_number cleaner0 2023-09-18T14:06:33Z DUMMY: Ala137 0.99882287 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.65771174 site cleaner0 2023-09-18T14:00:33Z SO: −4 bond_interaction MESH: cleaner0 2023-09-18T14:06:50Z parallel apolar contacts 0.99949133 residue_name_number cleaner0 2023-09-18T14:06:36Z DUMMY: Trp69 0.9983335 site cleaner0 2023-09-18T14:00:44Z SO: negative subsites 0.9988159 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9990707 protein cleaner0 2023-09-18T13:45:23Z PR: BaCel5A 0.9989895 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.9976179 protein_type cleaner0 2023-09-18T13:35:03Z MESH: xylanase 0.9923934 protein cleaner0 2023-09-18T13:45:27Z PR: CmXyn10B 0.99495846 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.9988996 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.99886346 protein_type cleaner0 2023-09-18T12:26:17Z MESH: cellulase 0.9983138 site cleaner0 2023-09-18T14:00:38Z SO: negative subsites 0.98889786 chemical cleaner0 2023-09-18T13:42:36Z CHEBI: sugars 0.98305774 protein_state cleaner0 2023-09-18T10:33:46Z DUMMY: bound in 0.9832241 site cleaner0 2023-09-18T14:00:48Z SO: −2 and −3 subsites 0.9674461 bond_interaction cleaner0 2023-09-18T12:26:11Z MESH: polar interactions 0.99883157 protein_type cleaner0 2023-09-18T10:31:50Z MESH: GH10 0.99209 protein_type cleaner0 2023-09-18T13:35:16Z MESH: xylanase 0.99729645 site cleaner0 2023-09-18T14:00:53Z SO: −2 subsite 0.99879384 protein_type cleaner0 2023-09-18T12:26:17Z MESH: cellulase RESULTS title_4 19682 The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity 0.98137945 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A RESULTS paragraph 19757 CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A. 0.9990533 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99842465 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.99872273 structure_element cleaner0 2023-09-18T10:36:12Z SO: CBMs 0.8389996 structure_element cleaner0 2023-09-18T10:36:40Z SO: CtCBM6 0.9042827 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 0.9175341 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.9989512 structure_element cleaner0 2023-09-18T13:51:47Z SO: fibronectin domain 0.99350643 structure_element cleaner0 2023-09-18T13:45:53Z SO: CtFn3 0.9994 structure_element cleaner0 2023-09-18T10:51:34Z SO: CBM6 0.93146616 protein_state cleaner0 2023-09-18T10:33:46Z DUMMY: bound in 0.9521939 protein_state cleaner0 2023-09-18T13:36:32Z DUMMY: exo-mode 0.986774 chemical cleaner0 2023-09-18T13:42:44Z CHEBI: xylo- and cellulooligosaccharides 0.99714065 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.99528027 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.99527496 structure_element cleaner0 2023-09-18T13:51:52Z SO: non-catalytic modules 0.99911505 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A protein_state DUMMY: cleaner0 2023-09-18T14:08:05Z truncated 0.99889565 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.96990776 experimental_method cleaner0 2023-09-18T12:26:35Z MESH: removal of 0.7631579 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.99899715 chemical cleaner0 2023-09-18T10:45:23Z CHEBI: WAX 0.99888617 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.8609744 experimental_method cleaner0 2023-09-18T12:26:40Z MESH: deletion of 0.9990049 structure_element cleaner0 2023-09-18T10:38:04Z SO: Fn3 0.9974022 experimental_method cleaner0 2023-09-18T12:26:38Z MESH: Truncation 0.99084926 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 0.979066 structure_element cleaner0 2023-09-18T10:52:05Z SO: CBM13 0.9990688 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99915814 chemical cleaner0 2023-09-18T13:42:49Z CHEBI: mannose 0.9989704 chemical cleaner0 2023-09-18T13:42:52Z CHEBI: galactose 0.777462 chemical cleaner0 2023-09-18T13:42:54Z CHEBI: complex glycans 0.9620865 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 0.99735713 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.9991768 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9986268 experimental_method cleaner0 2023-09-18T12:26:43Z MESH: Binding studies 0.9760868 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 0.99847585 chemical cleaner0 2023-09-18T13:42:57Z CHEBI: glycans 0.99904245 chemical cleaner0 2023-09-18T10:45:24Z CHEBI: WAX 0.99910945 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.999185 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.9991738 chemical cleaner0 2023-09-18T13:43:00Z CHEBI: mannose 0.99903405 chemical cleaner0 2023-09-18T13:43:03Z CHEBI: galactose 0.99697894 chemical cleaner0 2023-09-18T12:26:57Z CHEBI: birchwood xylan 0.9985886 chemical cleaner0 2023-09-18T13:43:07Z CHEBI: BX 0.9839033 structure_element cleaner0 2023-09-18T10:37:04Z SO: CtCBM13 0.99899536 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A RESULTS title_4 21061 Crystal Structure of CtXyl5A-D 0.99862117 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: Crystal Structure 0.9985922 mutant cleaner0 2023-09-18T12:27:42Z MESH: CtXyl5A-D RESULTS paragraph 21092 To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously. 0.99891555 structure_element cleaner0 2023-09-18T13:51:56Z SO: non-catalytic modules 0.99892277 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99893296 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: crystal structure 0.9983997 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.7888786 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.4889835 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.94939154 experimental_method cleaner0 2023-09-18T14:05:54Z MESH: crystallized 0.9952478 protein_state cleaner0 2023-09-18T13:36:36Z DUMMY: without structure_element SO: cleaner0 2023-09-18T10:38:10Z dockerin 0.9989806 mutant cleaner0 2023-09-18T12:27:42Z MESH: CtXyl5A-D 0.9989059 evidence cleaner0 2023-09-18T10:25:49Z DUMMY: crystal structure 0.9990243 protein_type cleaner0 2023-09-18T10:25:57Z MESH: arabinoxylanase 0.99872184 experimental_method cleaner0 2023-09-18T14:06:00Z MESH: molecular replacement 0.9985116 evidence cleaner0 2023-09-18T12:28:04Z DUMMY: Rwork 0.99814725 evidence cleaner0 2023-09-18T12:28:06Z DUMMY: Rfree 0.99822944 evidence cleaner0 2023-09-18T12:27:58Z DUMMY: structure 0.967706 residue_range cleaner0 2023-09-18T12:28:13Z DUMMY: Ala36 to Trp742 structure_element SO: cleaner0 2023-09-18T14:31:31Z GH5-CBM6-CBM13-Fn3 0.998767 evidence cleaner0 2023-09-18T12:27:52Z DUMMY: electron density 0.86748755 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 evidence DUMMY: cleaner0 2023-09-18T14:01:19Z crystal packing 0.9981135 site cleaner0 2023-09-18T14:01:00Z SO: solvent channel 0.61647874 structure_element cleaner0 2023-09-18T13:52:00Z SO: CBM62 0.99834853 evidence cleaner0 2023-09-18T12:28:08Z DUMMY: electron density 0.85926026 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.8196963 protein_state cleaner0 2023-09-18T13:36:40Z DUMMY: mobile 0.9980434 evidence cleaner0 2023-09-18T12:27:55Z DUMMY: structures 0.9983726 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.9979448 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 zbc0441653440005.jpg F5 FIG fig_caption 22184 Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks. 0.9992005 protein_type cleaner0 2023-09-18T10:25:58Z MESH: arabinoxylanase 0.99799275 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.9989273 structure_element cleaner0 2023-09-18T12:34:53Z SO: loop 0.9980647 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.9991582 structure_element cleaner0 2023-09-18T10:26:20Z SO: catalytic domain 0.9944258 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.9965469 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99827754 structure_element cleaner0 2023-09-18T13:52:05Z SO: fibronectin domain 0.99751484 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.99883205 structure_element cleaner0 2023-09-18T13:52:09Z SO: loop 0.9829196 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.9829951 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99495596 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds RESULTS paragraph 22746 CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v). 0.9912047 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.79160744 residue_range cleaner0 2023-09-18T12:29:19Z DUMMY: Gly567 to Pro648 protein_type MESH: cleaner0 2023-09-18T12:29:04Z CBM13 0.97495085 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99881685 structure_element cleaner0 2023-09-18T12:32:18Z SO: β-trefoil fold 0.99027336 structure_element cleaner0 2023-09-18T13:52:13Z SO: 3-fold repeating unit 0.78913796 residue_range cleaner0 2023-09-18T12:29:24Z DUMMY: 40–50 amino acid 0.998422 protein_type cleaner0 2023-09-18T13:35:21Z MESH: Ricin superfamily 0.8408495 structure_element cleaner0 2023-09-18T13:52:19Z SO: repeat 0.9983705 structure_element cleaner0 2023-09-18T12:32:15Z SO: antiparallel β-strands 0.9987341 experimental_method cleaner0 2023-09-18T14:06:09Z MESH: Dali search protein_type MESH: cleaner0 2023-09-18T12:29:39Z CBM13 0.998098 evidence cleaner0 2023-09-18T12:31:37Z DUMMY: root mean square deviation 0.99859744 species cleaner0 2023-09-18T10:38:26Z MESH: C. thermocellum protein_type MESH: cleaner0 2023-09-18T12:30:06Z exo-β-1,3-galactanase 0.9984226 species cleaner0 2023-09-18T12:30:13Z MESH: Streptomyces avermitilis protein_type MESH: cleaner0 2023-09-18T12:30:39Z β-l-arabinopyranosidase 0.9983331 species cleaner0 2023-09-18T12:30:17Z MESH: Streptomyces lividans 0.8736156 protein cleaner0 2023-09-18T13:47:34Z PR: xylanase 10A species MESH: cleaner0 2023-09-18T13:49:01Z Streptomyces olivaceoviridis E-86 0.7191514 protein cleaner0 2023-09-18T13:49:07Z PR: xylanase 10A RESULTS paragraph 23509 The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below). structure_element SO: cleaner0 2023-09-18T10:38:04Z Fn3 0.9970993 structure_element cleaner0 2023-09-18T12:32:51Z SO: β-sandwich fold structure_element SO: cleaner0 2023-09-18T13:52:42Z sheets 0.9945593 structure_element cleaner0 2023-09-18T13:52:48Z SO: antiparallel strands 0.99866545 structure_element cleaner0 2023-09-18T12:32:37Z SO: β1-β2-β5 0.9991293 structure_element cleaner0 2023-09-18T12:32:40Z SO: β-sheet 1 0.9986183 structure_element cleaner0 2023-09-18T12:32:42Z SO: β4-β3-β6 0.99922734 structure_element cleaner0 2023-09-18T12:32:44Z SO: β-sheet 2 0.999304 structure_element cleaner0 2023-09-18T12:32:47Z SO: β-sheet 2 0.6592578 site cleaner0 2023-09-18T14:01:28Z SO: cleft 0.732665 protein_type cleaner0 2023-09-18T12:31:58Z MESH: endo-binding CBMs 0.9990554 protein_state cleaner0 2023-09-18T13:07:19Z DUMMY: full-length 0.66877615 protein cleaner0 2023-09-18T13:49:11Z PR: enzyme 0.9982305 site cleaner0 2023-09-18T14:01:38Z SO: cleft 0.998874 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.9986582 chemical cleaner0 2023-09-18T10:28:43Z CHEBI: polysaccharide RESULTS paragraph 24045 In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13. 0.99554837 evidence cleaner0 2023-09-18T13:51:06Z DUMMY: structure 0.99779385 mutant cleaner0 2023-09-18T12:27:42Z MESH: CtXyl5A-D 0.7459186 structure_element cleaner0 2023-09-18T13:52:53Z SO: modules 0.9990779 site cleaner0 2023-09-18T14:01:43Z SO: interfaces structure_element SO: cleaner0 2023-09-18T14:32:00Z CtGH5-CBM6-CBM13 0.9977719 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.99530184 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 site SO: cleaner0 2023-09-18T14:02:09Z apolar solvent-exposed surface 0.9960426 bond_interaction cleaner0 2023-09-18T12:26:11Z MESH: polar interactions 0.99697936 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds 0.9962404 bond_interaction cleaner0 2023-09-18T14:10:23Z MESH: salt bridges bond_interaction MESH: cleaner0 2023-09-18T14:02:27Z apolar and polar interactions 0.9987067 protein_type cleaner0 2023-09-18T10:30:28Z MESH: glycoside hydrolases 0.99762887 structure_element cleaner0 2023-09-18T10:36:12Z SO: CBMs 0.9972363 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99929976 structure_element cleaner0 2023-09-18T12:34:12Z SO: central domain protein_state DUMMY: cleaner0 2023-09-18T13:37:21Z interacts with 0.9982716 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.9980007 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.998733 structure_element cleaner0 2023-09-18T12:33:43Z SO: CtFn3 0.99688613 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds 0.99871945 protein_state cleaner0 2023-09-18T13:37:41Z DUMMY: compact 0.99666494 oligomeric_state cleaner0 2023-09-18T13:37:35Z DUMMY: heterotetramer 0.9980378 site cleaner0 2023-09-18T12:33:28Z SO: CtCBM6-CBM13 interface 0.9993907 structure_element cleaner0 2023-09-18T12:34:15Z SO: linker 0.99948835 structure_element cleaner0 2023-09-18T13:06:17Z SO: SPISTGTIP 0.9606576 structure_element cleaner0 2023-09-18T13:52:56Z SO: modules 0.99290574 residue_name_number cleaner0 2023-09-18T14:08:18Z DUMMY: Ser514 0.9987361 residue_name_number cleaner0 2023-09-18T14:08:21Z DUMMY: Pro522 0.88606876 protein_state cleaner0 2023-09-18T13:37:47Z DUMMY: fixed conformation 0.9965569 residue_name cleaner0 2023-09-18T12:34:08Z SO: Ile bond_interaction MESH: cleaner0 2023-09-18T14:03:28Z apolar contacts 0.9991703 structure_element cleaner0 2023-09-18T12:34:48Z SO: linker 0.9988801 structure_element cleaner0 2023-09-18T10:36:12Z SO: CBMs 0.99847704 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.99815255 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.99904627 structure_element cleaner0 2023-09-18T12:33:53Z SO: loop 0.99917275 structure_element cleaner0 2023-09-18T12:33:59Z SO: β-7 0.99806833 structure_element cleaner0 2023-09-18T12:34:01Z SO: α-7 0.9991961 structure_element cleaner0 2023-09-18T12:33:56Z SO: loop 7 0.9988186 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 structure_element SO: cleaner0 2023-09-18T13:05:50Z trimodular clover 0.5572531 structure_element cleaner0 2023-09-18T13:53:02Z SO: modules 0.99958736 residue_name_number cleaner0 2023-09-18T12:34:40Z DUMMY: Trp285 bond_interaction MESH: cleaner0 2023-09-18T14:08:51Z intercalated between 0.99859947 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.997342 bond_interaction cleaner0 2023-09-18T12:19:58Z MESH: hydrogen bonds 0.99960214 residue_name_number cleaner0 2023-09-18T12:34:35Z DUMMY: Val615 0.9995946 residue_name_number cleaner0 2023-09-18T12:34:37Z DUMMY: Gly616 0.9986933 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 bond_interaction MESH: cleaner0 2023-09-18T14:02:58Z apolar contacts 0.9936412 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.99960405 residue_name_number cleaner0 2023-09-18T12:34:25Z DUMMY: Pro440 0.99960774 residue_name_number cleaner0 2023-09-18T12:34:28Z DUMMY: Phe489 0.9996075 residue_name_number cleaner0 2023-09-18T12:34:30Z DUMMY: Gly491 0.99959916 residue_name_number cleaner0 2023-09-18T12:34:32Z DUMMY: Ala492 0.99917793 structure_element cleaner0 2023-09-18T13:53:05Z SO: loop 7 0.98367256 protein_state cleaner0 2023-09-18T13:37:51Z DUMMY: completely disordered 0.9988607 protein_state cleaner0 2023-09-18T13:37:55Z DUMMY: truncated 0.69755924 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9978527 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.9964631 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.99774665 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.9942677 structure_element cleaner0 2023-09-18T12:34:23Z SO: loop 0.9990918 structure_element cleaner0 2023-09-18T12:34:18Z SO: loop 7 0.9989008 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.9995603 residue_name_number cleaner0 2023-09-18T12:34:43Z DUMMY: Glu279 0.9958968 structure_element cleaner0 2023-09-18T12:34:20Z SO: loop 0.5024981 experimental_method cleaner0 2023-09-18T12:34:46Z MESH: removal 0.99695325 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 experimental_method MESH: cleaner0 2023-09-18T14:04:06Z deletion structure_element SO: cleaner0 2023-09-18T10:37:05Z CtCBM13 RESULTS paragraph 26451 Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates. 0.96206796 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.983107 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99704546 bond_interaction cleaner0 2023-09-18T12:25:53Z MESH: hydrophobic interactions 0.994769 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99780685 structure_element cleaner0 2023-09-18T12:35:31Z SO: CtFn3 0.7309727 structure_element cleaner0 2023-09-18T13:53:10Z SO: modules 0.9990598 protein_state cleaner0 2023-09-18T13:07:30Z DUMMY: absence of 0.9950388 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 0.997883 evidence cleaner0 2023-09-18T12:35:39Z DUMMY: structure 0.9134587 structure_element cleaner0 2023-09-18T13:53:13Z SO: module 0.9861158 structure_element cleaner0 2023-09-18T10:37:36Z SO: CtCBM62 protein_state DUMMY: cleaner0 2023-09-18T14:05:12Z binding to 0.99920434 chemical cleaner0 2023-09-18T12:35:15Z CHEBI: d-Galp 0.99922657 chemical cleaner0 2023-09-18T13:43:19Z CHEBI: l-Arap 0.998511 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.99731666 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: Xylans 0.9827069 chemical cleaner0 2023-09-18T13:43:11Z CHEBI: sugars 0.9991762 chemical cleaner0 2023-09-18T12:35:15Z CHEBI: d-Galp 0.99790454 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.99599224 taxonomy_domain cleaner0 2023-09-18T10:43:47Z DUMMY: cereal 0.81789005 taxonomy_domain cleaner0 2023-09-18T12:35:51Z DUMMY: eucalyptus trees 0.99418384 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99656916 structure_element cleaner0 2023-09-18T10:37:37Z SO: CtCBM62 0.99837226 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9991806 chemical cleaner0 2023-09-18T12:35:15Z CHEBI: d-Galp 0.9913663 protein_state cleaner0 2023-09-18T13:06:30Z DUMMY: open 0.9990905 site cleaner0 2023-09-18T12:36:11Z SO: substrate binding cleft 0.9991543 protein_type cleaner0 2023-09-18T10:25:58Z MESH: arabinoxylanase chemical CHEBI: cleaner0 2023-09-18T10:29:26Z hemicellulose 0.99875224 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.998137 chemical cleaner0 2023-09-18T13:43:24Z CHEBI: glycans 0.99851733 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.8947423 structure_element cleaner0 2023-09-18T13:53:17Z SO: CBM62 RESULTS title_4 27651 Exploring GH5 Subfamily 34 0.9395437 protein_type cleaner0 2023-09-18T12:36:02Z MESH: GH5 Subfamily 34 RESULTS paragraph 27678 CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes. 0.99605703 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.99675363 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 protein_type MESH: cleaner0 2023-09-18T12:36:53Z GH5_34 0.9986108 species cleaner0 2023-09-18T10:38:26Z MESH: C. thermocellum protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.9954992 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.91863054 protein cleaner0 2023-09-18T10:50:32Z PR: AcGH5 0.9906282 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.94165695 structure_element cleaner0 2023-09-18T12:40:34Z SO: carbohydrate esterase family 6 module protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.98600787 taxonomy_domain cleaner0 2023-09-18T12:37:34Z DUMMY: Verrucomicrobiae 0.7465725 taxonomy_domain cleaner0 2023-09-18T12:37:44Z DUMMY: bacterium 0.993269 protein cleaner0 2023-09-18T10:49:41Z PR: VbGH5 structure_element SO: cleaner0 2023-09-18T14:32:27Z GH5-CBM6-CBM13 structure_element SO: cleaner0 2023-09-18T14:32:42Z Fn3-CBM62-dockerin 0.99669635 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A structure_element SO: cleaner0 2023-09-18T12:40:55Z Laminin_3_G domain 0.9380174 chemical cleaner0 2023-09-18T13:43:28Z CHEBI: carbohydrates 0.88023144 chemical cleaner0 2023-09-18T13:43:30Z CHEBI: glycan 0.63634735 taxonomy_domain cleaner0 2023-09-18T13:44:40Z DUMMY: Verrucomicobiae 0.77029544 protein_type cleaner0 2023-09-18T12:40:20Z MESH: GH43 subfamily 10 protein_type MESH: cleaner0 2023-09-18T12:42:40Z GH43_10 0.9972124 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.99884367 taxonomy_domain cleaner0 2023-09-18T12:38:21Z DUMMY: fungal protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.9986064 protein cleaner0 2023-09-18T12:47:40Z PR: GpGH5 0.99877983 taxonomy_domain cleaner0 2023-09-18T12:37:50Z DUMMY: bacterial 0.5396774 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.9984396 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.9987802 protein cleaner0 2023-09-18T12:47:40Z PR: GpGh5 0.99830353 species cleaner0 2023-09-18T12:37:55Z MESH: Gonapodya prolifera 0.99860615 taxonomy_domain cleaner0 2023-09-18T13:44:45Z DUMMY: fungus 0.99872905 taxonomy_domain cleaner0 2023-09-18T12:38:21Z DUMMY: fungal protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.99848294 species cleaner0 2023-09-18T12:38:02Z MESH: G. prolifera 0.9940562 taxonomy_domain cleaner0 2023-09-18T12:38:46Z DUMMY: Clostridium protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.9978834 species cleaner0 2023-09-18T12:38:02Z MESH: G. prolifera 0.8770961 taxonomy_domain cleaner0 2023-09-18T12:38:46Z DUMMY: Clostridium protein PR: cleaner0 2023-09-18T13:49:41Z GpGH5_34 0.9941837 taxonomy_domain cleaner0 2023-09-18T12:38:46Z DUMMY: Clostridium 0.99882096 taxonomy_domain cleaner0 2023-09-18T12:38:21Z DUMMY: fungal protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.9980454 structure_element cleaner0 2023-09-18T13:53:21Z SO: catalytic modules 0.9969531 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.99737984 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.9991984 chemical cleaner0 2023-09-18T10:45:28Z CHEBI: RAX 0.9991404 chemical cleaner0 2023-09-18T10:45:24Z CHEBI: WAX 0.9990416 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX 0.9986411 chemical cleaner0 2023-09-18T13:43:35Z CHEBI: BX 0.9990243 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases 0.98134685 protein cleaner0 2023-09-18T10:25:31Z PR: CtXyl5A 0.9585189 protein cleaner0 2023-09-18T10:50:32Z PR: AcGH5 0.9965377 protein cleaner0 2023-09-18T12:47:40Z PR: GpGH5 0.9943236 chemical cleaner0 2023-09-18T11:03:41Z CHEBI: oligosaccharides 0.99477345 chemical cleaner0 2023-09-18T11:03:41Z CHEBI: oligosaccharides 0.9852911 protein cleaner0 2023-09-18T10:49:42Z PR: VbGH5 0.9989858 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9989502 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases protein_type MESH: cleaner0 2023-09-18T12:42:40Z GH43_10 protein_type MESH: cleaner0 2023-09-18T10:54:58Z arabinofuranosidase protein_type MESH: cleaner0 2023-09-18T12:42:23Z GH43 chemical CHEBI: cleaner0 2023-09-18T10:26:42Z arabinose protein PR: cleaner0 2023-09-18T10:49:42Z VbGH5 structure_element SO: cleaner0 2023-09-18T10:35:55Z catalytic module protein PR: cleaner0 2023-09-18T10:50:32Z AcGH5 protein PR: cleaner0 2023-09-18T10:25:31Z CtXyl5A chemical CHEBI: cleaner0 2023-09-18T10:45:24Z WAX chemical CHEBI: cleaner0 2023-09-18T10:45:28Z RAX chemical CHEBI: cleaner0 2023-09-18T10:46:39Z CX protein_state DUMMY: cleaner0 2023-09-18T10:49:27Z wild type protein PR: cleaner0 2023-09-18T10:49:42Z VbGH5 protein_type MESH: cleaner0 2023-09-18T12:42:40Z GH43_10 chemical CHEBI: cleaner0 2023-09-18T10:26:42Z arabinose chemical CHEBI: cleaner0 2023-09-18T10:25:42Z arabinoxylans protein_type MESH: cleaner0 2023-09-18T10:25:58Z arabinoxylanase protein_state DUMMY: cleaner0 2023-09-18T12:44:44Z conserved residue_name_number DUMMY: cleaner0 2023-09-18T12:44:04Z Asp45 structure_element SO: cleaner0 2023-09-18T12:45:12Z GH43_10 protein PR: cleaner0 2023-09-18T10:49:42Z VbGH5 experimental_method MESH: cleaner0 2023-09-18T12:44:57Z substituted with residue_name SO: cleaner0 2023-09-18T12:44:24Z alanine structure_element SO: cleaner0 2023-09-18T10:35:55Z catalytic module mutant MESH: cleaner0 2023-09-18T10:49:23Z D45A protein_state DUMMY: cleaner0 2023-09-18T12:45:25Z mutant chemical CHEBI: cleaner0 2023-09-18T10:26:42Z arabinose protein_type MESH: cleaner0 2023-09-18T10:54:58Z arabinofuranosidase structure_element SO: cleaner0 2023-09-18T12:43:05Z GH43_10 protein_state DUMMY: cleaner0 2023-09-18T10:49:27Z wild type evidence DUMMY: cleaner0 2023-09-18T12:45:51Z kinetics protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 protein_type MESH: cleaner0 2023-09-18T10:25:58Z arabinoxylanase structure_element SO: cleaner0 2023-09-18T10:35:55Z catalytic module protein PR: cleaner0 2023-09-18T10:25:31Z CtXyl5A taxonomy_domain DUMMY: cleaner0 2023-09-18T12:38:21Z fungal protein_type MESH: cleaner0 2023-09-18T10:25:58Z arabinoxylanase chemical CHEBI: cleaner0 2023-09-18T10:45:24Z WAX chemical CHEBI: cleaner0 2023-09-18T10:45:28Z RAX protein PR: cleaner0 2023-09-18T10:25:32Z CtXyl5A taxonomy_domain DUMMY: cleaner0 2023-09-18T12:46:09Z eukaryotic taxonomy_domain DUMMY: cleaner0 2023-09-18T12:46:19Z prokaryotic chemical CHEBI: cleaner0 2023-09-18T10:46:39Z CX protein PR: cleaner0 2023-09-18T10:50:32Z AcGH5 protein PR: cleaner0 2023-09-18T10:49:42Z VbGH5 protein_state DUMMY: cleaner0 2023-09-18T13:07:19Z full-length zbc0441653440006.jpg F6 FIG fig_caption 31787 Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization. protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.93129015 experimental_method cleaner0 2023-09-18T12:46:59Z MESH: incubated 0.99875844 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9989768 protein cleaner0 2023-09-18T12:47:40Z PR: GpGH5 0.9987948 protein cleaner0 2023-09-18T10:49:42Z PR: VbGH5 0.9988304 protein cleaner0 2023-09-18T10:50:32Z PR: AcGH5 0.9960063 experimental_method cleaner0 2023-09-18T12:47:03Z MESH: TLC 0.99918824 chemical cleaner0 2023-09-18T13:43:43Z CHEBI: xylooligosaccharide DISCUSS title_1 32125 Discussion DISCUSS paragraph 32136 A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B. 0.99862134 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.9987355 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.99867666 structure_element cleaner0 2023-09-18T13:53:26Z SO: catalytic modules 0.865277 structure_element cleaner0 2023-09-18T13:53:30Z SO: flexible linker sequences 0.5641079 protein cleaner0 2023-09-18T10:25:32Z PR: CtXyl5A 0.99627954 evidence cleaner0 2023-09-18T13:51:11Z DUMMY: structure 0.99776864 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.86189497 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.95701545 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99906987 protein_state cleaner0 2023-09-18T13:38:00Z DUMMY: active 0.9981731 structure_element cleaner0 2023-09-18T10:35:55Z SO: catalytic module 0.9962853 structure_element cleaner0 2023-09-18T10:36:02Z SO: CtGH5 0.99705863 evidence cleaner0 2023-09-18T13:51:15Z DUMMY: crystallographic data 0.998901 chemical cleaner0 2023-09-18T13:43:49Z CHEBI: glycans 0.9889486 structure_element cleaner0 2023-09-18T10:37:05Z SO: CtCBM13 0.99855596 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan 0.99709713 chemical cleaner0 2023-09-18T13:43:53Z CHEBI: cellulose 0.9898572 structure_element cleaner0 2023-09-18T10:36:41Z SO: CtCBM6 0.9853876 chemical cleaner0 2023-09-18T13:43:56Z CHEBI: glycan 0.99907523 protein_state cleaner0 2023-09-18T13:07:18Z DUMMY: full-length 0.9986217 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant 0.9963482 structure_element cleaner0 2023-09-18T13:53:35Z SO: CBM46 0.9411532 taxonomy_domain cleaner0 2023-09-18T12:47:23Z DUMMY: Bacillus 0.99831474 protein_type cleaner0 2023-09-18T13:35:26Z MESH: xyloglucanase 0.9972825 protein_type cleaner0 2023-09-18T13:35:29Z MESH: mixed linked glucanase 0.9992636 protein cleaner0 2023-09-18T12:47:30Z PR: BhCel5B DISCUSS paragraph 33233 CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation. 0.9979913 protein cleaner0 2023-09-18T10:25:32Z PR: CtXyl5A 0.94887006 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 0.9977837 protein cleaner0 2023-09-18T10:25:32Z PR: CtXyl5A protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.99929583 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.99110305 site cleaner0 2023-09-18T14:04:24Z SO: specificity determinants 0.99954575 residue_name_number cleaner0 2023-09-18T10:26:54Z DUMMY: Glu68 0.9995704 residue_name_number cleaner0 2023-09-18T10:26:59Z DUMMY: Tyr92 0.99954885 residue_name_number cleaner0 2023-09-18T10:27:05Z DUMMY: Asn139 0.9985304 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.99864835 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9986283 site cleaner0 2023-09-18T14:04:29Z SO: −2* subsite 0.9983754 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.99908876 site cleaner0 2023-09-18T10:27:57Z SO: active site 0.99881303 structure_element cleaner0 2023-09-18T13:53:39Z SO: CBM62 0.9981012 protein cleaner0 2023-09-18T10:25:32Z PR: CtXyl5A 0.9863192 protein cleaner0 2023-09-18T10:50:32Z PR: AcGH5 0.99910295 chemical cleaner0 2023-09-18T10:24:32Z CHEBI: xylans 0.9990118 chemical cleaner0 2023-09-18T13:44:04Z CHEBI: d-galactose 0.70076585 protein_state cleaner0 2023-09-18T13:07:29Z DUMMY: absence of 0.9968041 structure_element cleaner0 2023-09-18T13:53:44Z SO: CBM 0.9990013 protein cleaner0 2023-09-18T12:47:40Z PR: GpGH5 0.99921906 protein_type cleaner0 2023-09-18T10:25:58Z MESH: arabinoxylanase 0.9991093 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.9977469 taxonomy_domain cleaner0 2023-09-18T13:44:50Z DUMMY: cereals protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 0.9990747 protein_type cleaner0 2023-09-18T13:35:33Z MESH: GHs 0.97536546 protein_type cleaner0 2023-09-18T13:49:58Z MESH: GH43 0.993919 protein_type cleaner0 2023-09-18T10:40:58Z MESH: GH5 protein_type MESH: cleaner0 2023-09-18T12:36:54Z GH5_34 DISCUSS paragraph 34568 An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX). 0.9990096 protein cleaner0 2023-09-18T10:49:42Z PR: VbGH5 0.9992551 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases 0.9983157 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.999131 protein cleaner0 2023-09-18T10:49:42Z PR: VbGH5 0.9985896 chemical cleaner0 2023-09-18T10:24:33Z CHEBI: xylans 0.9986557 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.9989998 chemical cleaner0 2023-09-18T10:25:42Z CHEBI: arabinoxylans 0.99906427 protein_type cleaner0 2023-09-18T13:35:38Z MESH: GH98 0.9987771 protein_type cleaner0 2023-09-18T13:35:42Z MESH: xylanase 0.9987264 chemical cleaner0 2023-09-18T10:24:33Z CHEBI: xylans chemical CHEBI: cleaner0 2023-09-18T10:46:39Z CX DISCUSS paragraph 35152 To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate. 0.9992999 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases 0.99896705 site cleaner0 2023-09-18T14:05:33Z SO: pocket 0.99837506 chemical cleaner0 2023-09-18T10:26:42Z CHEBI: arabinose 0.99813867 chemical cleaner0 2023-09-18T10:26:34Z CHEBI: xylose 0.9551583 protein_state cleaner0 2023-09-18T13:38:17Z DUMMY: open 0.99909574 site cleaner0 2023-09-18T12:49:02Z SO: xylan binding cleft 0.9953093 chemical cleaner0 2023-09-18T10:29:26Z CHEBI: hemicellulose 0.9972641 structure_element cleaner0 2023-09-18T13:53:50Z SO: catalytic modules 0.99897206 structure_element cleaner0 2023-09-18T10:36:13Z SO: CBMs 0.9993069 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases 0.9992654 protein_type cleaner0 2023-09-18T10:24:24Z MESH: arabinoxylanases 0.9987754 protein_type cleaner0 2023-09-18T10:35:36Z MESH: endo-xylanases 0.9984994 taxonomy_domain cleaner0 2023-09-18T10:24:42Z DUMMY: plant METHODS title_1 35932 Experimental Procedures METHODS title_4 35956 Cloning, Expression, and Purification of Components of CtXyl5A METHODS paragraph 36019 All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1. METHODS paragraph 37237 To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to >90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol. METHODS title_4 38285 Enzyme Assays METHODS paragraph 38299 CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously. METHODS title_4 39304 Oligosaccharide Analysis METHODS paragraph 39329 Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously METHODS title_4 39853 Crystallography METHODS paragraph 39869 Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot). METHODS paragraph 41279 To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2. T2.xml T2 TABLE table_caption 43158 Data collection and refinement statistics evidence DUMMY: cleaner0 2023-09-18T12:51:14Z Data collection and refinement statistics T2.xml T2 TABLE table_caption 43200 The values in parentheses are for highest resolution shell. T2.xml T2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead valign="bottom"><tr><th rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">CtXyl5A_-D</th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Arap</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Xylp</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6- (<italic>Araf</italic>-Xyl<italic>p</italic>₄)</th></tr></thead><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data collection</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    Source</td><td align="left" rowspan="1" colspan="1">ESRF-ID14-1</td><td align="left" rowspan="1" colspan="1">Diamond I04–1</td><td align="left" rowspan="1" colspan="1">Diamond I24</td><td align="left" rowspan="1" colspan="1">Diamond I02</td></tr><tr><td align="left" rowspan="1" colspan="1">    Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9334</td><td align="left" rowspan="1" colspan="1">0.9173</td><td align="left" rowspan="1" colspan="1">0.9772</td><td align="left" rowspan="1" colspan="1">0.9791</td></tr><tr><td align="left" rowspan="1" colspan="1">    Space group</td><td align="left" rowspan="1" colspan="1">P2₁2₁2</td><td align="left" rowspan="1" colspan="1">P2₁2₁2₁</td><td align="left" rowspan="1" colspan="1">P2₁2₁2₁</td><td align="left" rowspan="1" colspan="1">P2₁2₁2₁</td></tr><tr><td align="left" rowspan="1" colspan="1">    Cell dimensions</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">147.4, 191.7, 50.7</td><td align="left" rowspan="1" colspan="1">67.1, 72.4, 109.1</td><td align="left" rowspan="1" colspan="1">67.9, 72.5, 109.5</td><td align="left" rowspan="1" colspan="1">76.3, 123.2, 125.4</td></tr><tr><td align="left" rowspan="1" colspan="1">        α, β, γ (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. of measured reflections</td><td align="left" rowspan="1" colspan="1">244,475 (29,324)</td><td align="left" rowspan="1" colspan="1">224,842 (11,281)</td><td align="left" rowspan="1" colspan="1">152,004 (4,996)</td><td align="left" rowspan="1" colspan="1">463,237 (23,068)</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. of independent reflections</td><td align="left" rowspan="1" colspan="1">42246 (5,920)</td><td align="left" rowspan="1" colspan="1">63,523 (3,175)</td><td align="left" rowspan="1" colspan="1">42,716 (2,334)</td><td align="left" rowspan="1" colspan="1">140,288 (6,879)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Resolution (Å)</td><td align="left" rowspan="1" colspan="1">50.70–2.64 (2.78–2.64)</td><td align="left" rowspan="1" colspan="1">44.85–1.65 (1.68–1.65)</td><td align="left" rowspan="1" colspan="1">45.16–1.90 (1.94–1.90)</td><td align="left" rowspan="1" colspan="1">48.43–1.65 (1.68–1.65)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_merge (%)</td><td align="left" rowspan="1" colspan="1">16.5 (69.5)</td><td align="left" rowspan="1" colspan="1">6.7 (65.1)</td><td align="left" rowspan="1" colspan="1">2.8 (8.4)</td><td align="left" rowspan="1" colspan="1">5.7 (74.9)</td></tr><tr><td align="left" rowspan="1" colspan="1">    CC_1/2</td><td align="left" rowspan="1" colspan="1">0.985 (0.478)</td><td align="left" rowspan="1" colspan="1">0.998 (0.594)</td><td align="left" rowspan="1" colspan="1">0.999 (0.982)</td><td align="left" rowspan="1" colspan="1">0.998 (0.484)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>I</italic>/σ<italic>I</italic></td><td align="left" rowspan="1" colspan="1">8.0 (2.0)</td><td align="left" rowspan="1" colspan="1">13 (1.6)</td><td align="left" rowspan="1" colspan="1">26.6 (8.0)</td><td align="left" rowspan="1" colspan="1">11.2 (1.6)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Completeness (%)</td><td align="left" rowspan="1" colspan="1">98.5 (96.4)</td><td align="left" rowspan="1" colspan="1">98.5 (99.4)</td><td align="left" rowspan="1" colspan="1">98.6 (85.0)</td><td align="left" rowspan="1" colspan="1">98.8 (99.4)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Redundancy</td><td align="left" rowspan="1" colspan="1">5.8 (5.0)</td><td align="left" rowspan="1" colspan="1">3.5 (3.6)</td><td align="left" rowspan="1" colspan="1">3.6 (2.1)</td><td align="left" rowspan="1" colspan="1">3.3 (3.4)</td></tr><tr><td colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Refinement</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic>_work/<italic>R</italic>_free</td><td align="left" rowspan="1" colspan="1">23.7/27.8</td><td align="left" rowspan="1" colspan="1">12.2/17.0</td><td align="left" rowspan="1" colspan="1">12.9/16.1</td><td align="left" rowspan="1" colspan="1">14.5/19.9</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. atoms</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Protein</td><td align="left" rowspan="1" colspan="1">5446</td><td align="left" rowspan="1" colspan="1">3790</td><td align="left" rowspan="1" colspan="1">3729</td><td align="left" rowspan="1" colspan="1">7333</td></tr><tr><td align="left" rowspan="1" colspan="1">        Ligand</td><td align="left" rowspan="1" colspan="1">19</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">92</td></tr><tr><td align="left" rowspan="1" colspan="1">        Water</td><td align="left" rowspan="1" colspan="1">227</td><td align="left" rowspan="1" colspan="1">579</td><td align="left" rowspan="1" colspan="1">601</td><td align="left" rowspan="1" colspan="1">923</td></tr><tr><td align="left" rowspan="1" colspan="1">    B-factors</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Protein</td><td align="left" rowspan="1" colspan="1">41.6</td><td align="left" rowspan="1" colspan="1">17.8</td><td align="left" rowspan="1" colspan="1">15.8</td><td align="left" rowspan="1" colspan="1">21.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Ligand</td><td align="left" rowspan="1" colspan="1">65.0</td><td align="left" rowspan="1" colspan="1">19.4</td><td align="left" rowspan="1" colspan="1">24.2</td><td align="left" rowspan="1" colspan="1">39.5</td></tr><tr><td align="left" rowspan="1" colspan="1">        Water</td><td align="left" rowspan="1" colspan="1">35.4</td><td align="left" rowspan="1" colspan="1">38.5</td><td align="left" rowspan="1" colspan="1">32.2</td><td align="left" rowspan="1" colspan="1">37.6</td></tr><tr><td align="left" rowspan="1" colspan="1">    R.m.s deviations</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.008</td><td align="left" rowspan="1" colspan="1">0.015</td><td align="left" rowspan="1" colspan="1">0.012</td><td align="left" rowspan="1" colspan="1">0.012</td></tr><tr><td align="left" rowspan="1" colspan="1">        Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.233</td><td align="left" rowspan="1" colspan="1">1.502</td><td align="left" rowspan="1" colspan="1">1.624</td><td align="left" rowspan="1" colspan="1">1.554</td></tr><tr><td align="left" rowspan="1" colspan="1">    Protein Data Bank code</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5G56">5G56</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA0">5LA0</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA1">5LA1</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="2LA2">2LA2</ext-link></td></tr></tbody></table> 43260 CtXyl5A-D GH5-CBM6-Arap GH5-CBM6-Xylp GH5-CBM6- (Araf-Xylp4) Data collection     Source ESRF-ID14-1 Diamond I04–1 Diamond I24 Diamond I02     Wavelength (Å) 0.9334 0.9173 0.9772 0.9791     Space group P21212 P212121 P212121 P212121     Cell dimensions         a, b, c (Å) 147.4, 191.7, 50.7 67.1, 72.4, 109.1 67.9, 72.5, 109.5 76.3, 123.2, 125.4         α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90     No. of measured reflections 244,475 (29,324) 224,842 (11,281) 152,004 (4,996) 463,237 (23,068)     No. of independent reflections 42246 (5,920) 63,523 (3,175) 42,716 (2,334) 140,288 (6,879)     Resolution (Å) 50.70–2.64 (2.78–2.64) 44.85–1.65 (1.68–1.65) 45.16–1.90 (1.94–1.90) 48.43–1.65 (1.68–1.65)     Rmerge (%) 16.5 (69.5) 6.7 (65.1) 2.8 (8.4) 5.7 (74.9)     CC1/2 0.985 (0.478) 0.998 (0.594) 0.999 (0.982) 0.998 (0.484)     I/σI 8.0 (2.0) 13 (1.6) 26.6 (8.0) 11.2 (1.6)     Completeness (%) 98.5 (96.4) 98.5 (99.4) 98.6 (85.0) 98.8 (99.4)     Redundancy 5.8 (5.0) 3.5 (3.6) 3.6 (2.1) 3.3 (3.4) Refinement     Rwork/Rfree 23.7/27.8 12.2/17.0 12.9/16.1 14.5/19.9     No. atoms         Protein 5446 3790 3729 7333         Ligand 19 20 20 92         Water 227 579 601 923     B-factors         Protein 41.6 17.8 15.8 21.0         Ligand 65.0 19.4 24.2 39.5         Water 35.4 38.5 32.2 37.6     R.m.s deviations         Bond lengths (Å) 0.008 0.015 0.012 0.012         Bond angles (°) 1.233 1.502 1.624 1.554     Protein Data Bank code 5G56 5LA0 5LA1 2LA2 mutant MESH: cleaner0 2023-09-18T12:27:43Z CtXyl5A-D 0.9927343 complex_assembly cleaner0 2023-09-18T12:52:03Z GO: GH5-CBM6-Arap 0.99599856 complex_assembly cleaner0 2023-09-18T12:52:14Z GO: GH5-CBM6-Xylp complex_assembly GO: cleaner0 2023-09-18T12:52:48Z GH5-CBM6- (Araf-Xylp4) 0.91044104 evidence cleaner0 2023-09-18T12:52:52Z DUMMY: Rwork 0.8382509 evidence cleaner0 2023-09-18T12:52:54Z DUMMY: Rfree AUTH_CONT title_1 45145 Author Contributions AUTH_CONT paragraph 45166 A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper. SUPPL title_1 45944 Supplementary Material SUPPL footnote 45967 This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article. SUPPL footnote 46502 This article contains supplemental Table S1 and Fig. S1. SUPPL footnote 46559 GH 0.9930426 protein_type cleaner0 2023-09-18T10:31:28Z MESH: GH SUPPL footnote 46562 glycoside hydrolase 0.9983388 protein_type cleaner0 2023-09-18T10:31:20Z MESH: glycoside hydrolase SUPPL footnote 46582 CtXyl5A 0.9968766 protein cleaner0 2023-09-18T10:25:32Z PR: CtXyl5A SUPPL footnote 46590 C. thermocellum arabinoxylanase 0.9984484 species cleaner0 2023-09-18T10:38:26Z MESH: C. thermocellum 0.998863 protein_type cleaner0 2023-09-18T10:25:58Z MESH: arabinoxylanase SUPPL footnote 46622 CBM 0.99871993 structure_element cleaner0 2023-09-18T13:53:56Z SO: CBM SUPPL footnote 46626 non-catalytic carbohydrate binding module 0.82269233 structure_element cleaner0 2023-09-18T10:26:26Z SO: non-catalytic carbohydrate binding module SUPPL footnote 46668 Fn 0.40625653 protein_type cleaner0 2023-09-18T13:54:48Z MESH: Fn SUPPL footnote 46671 fibronectin 0.6230605 protein_type cleaner0 2023-09-18T13:35:49Z MESH: fibronectin SUPPL footnote 46683 WAX 0.9978181 chemical cleaner0 2023-09-18T10:45:24Z CHEBI: WAX SUPPL footnote 46687 wheat arabinoxylan 0.91318005 taxonomy_domain cleaner0 2023-09-18T10:44:28Z DUMMY: wheat 0.9990382 chemical cleaner0 2023-09-18T13:44:12Z CHEBI: arabinoxylan SUPPL footnote 46706 RAX 0.87364924 chemical cleaner0 2023-09-18T10:45:28Z CHEBI: RAX SUPPL footnote 46710 rye arabinoxylan 0.8627195 taxonomy_domain cleaner0 2023-09-18T10:44:42Z DUMMY: rye 0.99903655 chemical cleaner0 2023-09-18T13:44:15Z CHEBI: arabinoxylan SUPPL footnote 46727 CX 0.9888843 chemical cleaner0 2023-09-18T10:46:39Z CHEBI: CX SUPPL footnote 46730 corn bran xylan 0.6373213 taxonomy_domain cleaner0 2023-09-18T10:45:09Z DUMMY: corn 0.8507954 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan SUPPL footnote 46746 HPAEC 0.8287563 experimental_method cleaner0 2023-09-18T10:58:50Z MESH: HPAEC SUPPL footnote 46752 high performance anion exchange chromatography 0.99853724 experimental_method cleaner0 2023-09-18T14:06:16Z MESH: high performance anion exchange chromatography SUPPL footnote 46799 PDB SUPPL footnote 46803 Protein Data Bank SUPPL footnote 46821 BX SUPPL footnote 46824 birchwood xylan 0.96442974 taxonomy_domain cleaner0 2023-09-18T13:44:55Z DUMMY: birchwood 0.99584645 chemical cleaner0 2023-09-18T10:24:49Z CHEBI: xylan SUPPL footnote 46840 ESI SUPPL footnote 46844 electrospray ionization. 0.99849916 experimental_method cleaner0 2023-09-18T14:06:20Z MESH: electrospray ionization SUPPL footnote 46869 The abbreviations used are: REF title 46898 References 444 455 surname:Gilbert;given-names:H. J. 20406913 REF Plant Physiol ref 153 2010 46909 The biochemistry and structural biology of plant cell wall deconstruction 316 317 surname:Himmel;given-names:M. E. surname:Bayer;given-names:E. A. 19523813 REF Curr. Opin. Biotechnol ref 20 2009 46983 Lignocellulose conversion to biofuels: current challenges, global perspectives surname:Brett;given-names:C. W. surname:K. REF Physiology and biochemistry of plant cell walls: Topics in Plant Functional Biology ref 1996 47062 1 67 surname:Ebringerova;given-names:A. surname:Hromadkova;given-names:Z. surname:Heinze;given-names:T. surname:Hienzy;given-names:T. REF Polysaccharide I ref 2005 47063 Structure, characterization and use 6141 6148 surname:Agger;given-names:J. surname:Viksø-Nielsen;given-names:A. surname:Meyer;given-names:A. S. 20411987 REF J. Agric. Food Chem ref 58 2010 47099 Enzymatic xylose release from pretreated corn bran arabinoxylan: differential effects of deacetylation and deferuloylation on insoluble and soluble substrate fractions D490 D495 surname:Lombard;given-names:V. surname:Golaconda Ramulu;given-names:H. surname:Drula;given-names:E. surname:Coutinho;given-names:P. M. surname:Henrissat;given-names:B. 24270786 REF Nucleic. Acids Res ref 42 2014 47267 The carbohydrate-active enzymes database (CAZy) in 2013 338 348 surname:Gilbert;given-names:H. J. surname:Stålbrand;given-names:H. surname:Brumer;given-names:H. 18430603 REF Curr. Opin. Plant Biol ref 11 2008 47323 How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation 9597 9605 surname:Pell;given-names:G. surname:Taylor;given-names:E. J. surname:Gloster;given-names:T. M. surname:Turkenburg;given-names:J. P. surname:Fontes;given-names:C. M. surname:Ferreira;given-names:L. M. surname:Nagy;given-names:T. surname:Clark;given-names:S. J. surname:Davies;given-names:G. J. surname:Gilbert;given-names:H. J. 14668328 REF J. Biol. Chem ref 279 2004 47425 The mechanisms by which family 10 glycoside hydrolases bind decorated substrates 1666 1677 surname:Vrsanská;given-names:M. surname:Kolenová;given-names:K. surname:Puchart;given-names:V. surname:Biely;given-names:P. 17381510 REF FEBS J ref 274 2007 47506 Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi 557 559 surname:Davies;given-names:G. J. surname:Wilson;given-names:K. S. surname:Henrissat;given-names:B. 9020895 REF Biochem. J ref 321 1997 47610 Nomenclature for sugar-binding subsites in glycosyl hydrolases 22510 22520 surname:Correia;given-names:M. A. surname:Mazumder;given-names:K. surname:Brás;given-names:J. L. surname:Firbank;given-names:S. J. surname:Zhu;given-names:Y. surname:Lewis;given-names:R. J. surname:York;given-names:W. S. surname:Fontes;given-names:C. M. surname:Gilbert;given-names:H. J. 21378160 REF J. Biol. Chem ref 286 2011 47673 Structure and function of an arabinoxylan-specific xylanase 878 880 surname:Alahuhta;given-names:M. surname:Xu;given-names:Q. surname:Brunecky;given-names:R. surname:Adney;given-names:W. S. surname:Ding;given-names:S. Y. surname:Himmel;given-names:M. E. surname:Lunin;given-names:V. V. REF Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun ref 66 2010 47733 Structure of a fibronectin type III-like module from Clostridium thermocellum 521 554 surname:Bayer;given-names:E. A. surname:Belaich;given-names:J. P. surname:Shoham;given-names:Y. surname:Lamed;given-names:R. 15487947 REF Annu. Rev. Microbiol ref 58 2004 47811 The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides 655 681 surname:Fontes;given-names:C. M. surname:Gilbert;given-names:H. J. 20373916 REF Annu. Rev. Biochem ref 79 2010 47901 Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates 22499 22509 surname:Montanier;given-names:C. Y. surname:Correia;given-names:M. A. surname:Henrissat;given-names:B. surname:Fontes;given-names:C. M. surname:Gilbert;given-names:H. J. surname:Flint;given-names:J. E. surname:Zhu;given-names:Y. surname:Baslé;given-names:A. surname:McKee;given-names:L. S. surname:Prates;given-names:J. A. surname:Polizzi;given-names:S. J. surname:Coutinho;given-names:P. M. surname:Lewis;given-names:R. J. 21454512 REF J. Biol. Chem ref 286 2011 48007 A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization 1293 1305 surname:Vardakou;given-names:M. surname:Dumon;given-names:C. surname:Murray;given-names:J. W. surname:Christakopoulos;given-names:P. surname:Weiner;given-names:D. P. surname:Juge;given-names:N. surname:Lewis;given-names:R. J. surname:Gilbert;given-names:H. J. surname:Flint;given-names:J. E. 18078955 REF J. Mol. Biol ref 375 2008 48160 Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases 607 614 surname:Beylot;given-names:M. H. surname:McKie;given-names:V. A. surname:Voragen;given-names:A. G. surname:Doeswijk-Voragen;given-names:C. H. surname:Gilbert;given-names:H. J. 11535122 REF Biochem. J ref 358 2001 48264 The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity 2942 2951 surname:Charnock;given-names:S. J. surname:Lakey;given-names:J. H. surname:Virden;given-names:R. surname:Hughes;given-names:N. surname:Sinnott;given-names:M. L. surname:Hazlewood;given-names:G. P. surname:Pickersgill;given-names:R. surname:Gilbert;given-names:H. J. 9006940 REF J. Biol. Chem ref 272 1997 48376 Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan 32187 32199 surname:Charnock;given-names:S. J. surname:Spurway;given-names:T. D. surname:Xie;given-names:H. surname:Beylot;given-names:M. H. surname:Virden;given-names:R. surname:Warren;given-names:R. A. surname:Hazlewood;given-names:G. P. surname:Gilbert;given-names:H. J. 9822697 REF J. Biol. Chem ref 273 1998 48585 The topology of the substrate binding clefts of glycosyl hydrolase family 10 xylanases are not conserved 575 585 surname:Fujimoto;given-names:Z. surname:Kuno;given-names:A. surname:Kaneko;given-names:S. surname:Yoshida;given-names:S. surname:Kobayashi;given-names:H. surname:Kusakabe;given-names:I. surname:Mizuno;given-names:H. 10884353 REF J. Mol. Biol ref 300 2000 48690 Crystal structure of Streptomyces olivaceoviridis E-86 β-xylanase containing xylan-binding domain 25097 25106 surname:Ichinose;given-names:H. surname:Fujimoto;given-names:Z. surname:Honda;given-names:M. surname:Harazono;given-names:K. surname:Nishimoto;given-names:Y. surname:Uzura;given-names:A. surname:Kaneko;given-names:S. 19608743 REF J. Biol. Chem ref 284 2009 48791 A β-l-arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27 447 457 surname:Jiang;given-names:D. surname:Fan;given-names:J. surname:Wang;given-names:X. surname:Zhao;given-names:Y. surname:Huang;given-names:B. surname:Liu;given-names:J. surname:Zhang;given-names:X. C. 22960181 REF J. Struct. Biol ref 180 2012 48901 Crystal structure of 1,3Gal43A, an exo-beta-1,3-galactanase from Clostridium thermocellum 4246 4254 surname:Notenboom;given-names:V. surname:Boraston;given-names:A. B. surname:Williams;given-names:S. J. surname:Kilburn;given-names:D. G. surname:Rose;given-names:D. R. 11914070 REF Biochemistry ref 41 2002 48991 High-resolution crystal structures of the lectin-like xylan binding domain from Streptomyces lividans xylanase 10A with bound substrates reveal a novel mode of xylan binding 769 781 surname:Boraston;given-names:A. B. surname:Bolam;given-names:D. N. surname:Gilbert;given-names:H. J. surname:Davies;given-names:G. J. 15214846 REF Biochem. J ref 382 2004 49165 Carbohydrate-binding modules: fine-tuning polysaccharide recognition 669 677 surname:Gilbert;given-names:H. J. surname:Knox;given-names:J. P. surname:Boraston;given-names:A. B. 23769966 REF Curr. Opin. Struct. Biol ref 23 2013 49234 Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules 2091 2100 surname:Poon;given-names:D. K. surname:Withers;given-names:S. G. surname:McIntosh;given-names:L. P. 17121820 REF J. Biol. Chem ref 282 2007 49358 Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi xylanase Cex through NMR spectroscopic analysis 10572 10586 surname:Venditto;given-names:I. surname:Najmudin;given-names:S. surname:Luís;given-names:A. S. surname:Ferreira;given-names:L. M. surname:Sakka;given-names:K. surname:Knox;given-names:J. P. surname:Gilbert;given-names:H. J. surname:Fontes;given-names:C. M. 25713075 REF J. Biol. Chem ref 290 2015 49516 Family 46 carbohydrate-binding modules contribute to the enzymatic hydrolysis of xyloglucan and β-1,3–1,4-glucans through distinct mechanisms 15293 15298 surname:Hervé;given-names:C. surname:Rogowski;given-names:A. surname:Blake;given-names:A. W. surname:Marcus;given-names:S. E. surname:Gilbert;given-names:H. J. surname:Knox;given-names:J. P. 20696902 REF Proc. Natl. Acad. Sci. U.S.A ref 107 2010 49661 Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects 186 surname:Aspeborg;given-names:H. surname:Coutinho;given-names:P. M. surname:Wang;given-names:Y. surname:Brumer;given-names:H.;suffix:3rd surname:Henrissat;given-names:B. 22992189 REF BMC Evol. Biol ref 12 2012 49789 Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5) 165 169 surname:Cuskin;given-names:F. surname:Lowe;given-names:E. C. surname:Rogowski;given-names:A. surname:Hamilton;given-names:B. S. surname:Chen;given-names:R. surname:Tolbert;given-names:T. J. surname:Piens;given-names:K. surname:Temple;given-names:M. J. surname:Zhu;given-names:Y. surname:Cameron;given-names:E. A. surname:Pudlo;given-names:N. A. surname:Porter;given-names:N. T. surname:Urs;given-names:K. surname:Thompson;given-names:A. J. surname:Cartmell;given-names:A. 25567280 REF Nature ref 517 2015 49889 Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism 1686 1692 surname:Mewis;given-names:K. surname:Lenfant;given-names:N. surname:Lombard;given-names:V. surname:Henrissat;given-names:B. 26729713 REF Appl. Environ. Microbiol ref 82 2016 49966 Dividing the large glycoside hydrolase family 43 into subfamilies: a motivation for detailed enzyme characterization 12 21 surname:Chen;given-names:V. B. surname:Arendall;given-names:W. B.;suffix:3rd surname:Headd;given-names:J. J. surname:Keedy;given-names:D. A. surname:Immormino;given-names:R. M. surname:Kapral;given-names:G. J. surname:Murray;given-names:L. W. surname:Richardson;given-names:J. S. surname:Richardson;given-names:D. C. 20057044 REF Acta Crystallogr. D Biol. Crystallogr ref 66 2010 50083 MolProbity: all-atom structure validation for macromolecular crystallography 14077 14082 surname:Charnock;given-names:S. J. surname:Bolam;given-names:D. N. surname:Nurizzo;given-names:D. surname:Szabó;given-names:L. surname:McKie;given-names:V. A. surname:Gilbert;given-names:H. J. surname:Davies;given-names:G. J. 12391332 REF Proc. Natl. Acad. Sci. U.S.A ref 99 2002 50160 Promiscuity in ligand-binding: the three-dimensional structure of a Piromyces carbohydrate-binding module, CBM29–2, in complex with cello- and mannohexaose 426 428 surname:Miller;given-names:G. L. REF Anal. Chem ref 31 1959 50318 Use of dinitrosalicylic acid reagent for determination of reducing sugar 2697 2702 surname:Proctor;given-names:M. R. surname:Taylor;given-names:E. J. surname:Nurizzo;given-names:D. surname:Turkenburg;given-names:J. P. surname:Lloyd;given-names:R. M. surname:Vardakou;given-names:M. surname:Davies;given-names:G. J. surname:Gilbert;given-names:H. J. 15708971 REF Proc. Natl. Acad. Sci. U.S.A ref 102 2005 50391 Tailored catalysts for plant cell-wall degradation: Redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A 494 505 surname:Bui;given-names:N. K. surname:Gray;given-names:J. surname:Schwarz;given-names:H. surname:Schumann;given-names:P. surname:Blanot;given-names:D. surname:Vollmer;given-names:W. 18996994 REF J. Bacteriol ref 191 2009 50519 The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development surname:Leslie;given-names:A. G. W. surname:Powell;given-names:H. R. surname:Read;given-names:R. J. surname:Sussman;given-names:J. L. REF Evolving Methods for Macromolecular Crystallography ref 2007 50662 Processing diffraction data with mosflm 72 82 surname:Evans;given-names:P. 16369096 REF Acta Crystallogr. D Biol. Crystallogr ref 62 2006 50702 Scaling and assessment of data quality 833 836 surname:Brás;given-names:J. L. surname:Correia;given-names:M. A. surname:Romão;given-names:M. J. surname:Prates;given-names:J. A. surname:Fontes;given-names:C. M. surname:Najmudin;given-names:S. REF Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun ref 67 2011 50741 Purification, crystallization and preliminary x-ray characterization of the pentamodular arabinoxylanase CtXyl5A from Clostridium thermocellum 540 545 surname:Stierand;given-names:K. surname:Rarey;given-names:M. 24900245 REF ACS Med. Chem. Lett ref 1 2010 50884 Drawing the PDB: protein-ligand complexes in two dimensions 658 674 surname:McCoy;given-names:A. J. surname:Grosse-Kunstleve;given-names:R. W. surname:Adams;given-names:P. D. surname:Winn;given-names:M. D. surname:Storoni;given-names:L. C. surname:Read;given-names:R. J. 19461840 REF J. Appl. Crystallogr ref 40 2007 50944 Phaser crystallographic software 1002 1011 surname:Cowtan;given-names:K. 16929101 REF Acta Crystallogr. D Biol. Crystallogr ref 62 2006 50977 The Buccaneer software for automated model building: 1. Tracing protein chains 61 69 surname:Terwilliger;given-names:T. C. surname:Grosse-Kunstleve;given-names:R. W. surname:Afonine;given-names:P. V. surname:Moriarty;given-names:N. W. surname:Zwart;given-names:P. H. surname:Hung;given-names:L. W. surname:Read;given-names:R. J. surname:Adams;given-names:P. D. 18094468 REF Acta Crystallogr. D Biol. Crystallogr ref 64 2008 51056 Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard 2126 2132 surname:Emsley;given-names:P. surname:Cowtan;given-names:K. 15572765 REF Acta Crystallogr. D Biol. Crystallogr ref 60 2004 51161 Coot: model-building tools for molecular graphics 355 367 surname:Murshudov;given-names:G. N. surname:Skubák;given-names:P. surname:Lebedev;given-names:A. A. surname:Pannu;given-names:N. S. surname:Steiner;given-names:R. A. surname:Nicholls;given-names:R. A. surname:Winn;given-names:M. D. surname:Long;given-names:F. surname:Vagin;given-names:A. A. 21460454 REF Acta Crystallogr. D Biol. Crystallogr ref 67 2011 51211 REFMAC5 for the refinement of macromolecular crystal structures D355 D359 surname:Laskowski;given-names:R. A. 18996896 REF Nucleic Acids Res ref 37 2009 51275 PDBsum new things 15483 15495 surname:Cartmell;given-names:A. surname:McKee;given-names:L. S. surname:Marles-Wright;given-names:J. surname:Peña;given-names:M. J. surname:Larsbrink;given-names:J. surname:Brumer;given-names:H. surname:Kaneko;given-names:S. surname:Ichinose;given-names:H. surname:Lewis;given-names:R. J. surname:Viksø-Nielsen;given-names:A. surname:Gilbert;given-names:H. J. 21339299 REF J. Biol. Chem ref 286 2011 51293 The structure and function of an arabinan-specific α-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases 125 132 surname:Kabsch;given-names:W. 20124692 REF Acta Crystallogr D Biol. Crystallogr ref 66 2010 51454 XDS 1022 1025 surname:Vagin;given-names:A. surname:Teplyakov;given-names:A. REF J. Appl. Crystallogr ref 30 1997 51458 MolRep: an automated program for molecular replacement