bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autophosphorylation‐dependent inactivation of plant chimeric calcium/calmodulin‐dependent protein kinase
Authors: Sathyanarayanan P. V. , Poovaiah B. W. 
Published: March 2003

Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin‐like Ca2+‐binding domain unlike other known calmodulin‐	dependent kinases. Ca2+‐Binding to the visinin‐like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417–30422]. Here, we report that the Ca2+‐stimulated autophosphorylation of CCaMK results in time‐dependent loss of enzyme activity. This time‐dependent loss of activity or self‐inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self‐association. Specifically, autophosphorylation in the presence of 200 µm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild‐type kinase in the presence of AMP‐PNP (an unhydrolyzable ATP analog) or the autophosphorylation‐site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation‐ dependent self‐inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.
Expression and purification of the recombinant subunits of toluene/o‐xylene monooxygenase and reconstitution of the active complex
Authors: Cafaro Valeria , Scognamiglio Roberta , Viggiani Ambra , Izzo Viviana , Passaro Irene , Notomista Eugenio , Piaz Fabrizio Dal , Amoresano Angela , Casbarra Annarita , Pucci Piero , Di Donato Alberto 
Published: March 2003

This paper describes the cloning of the genes coding for each component of the complex of toluene/o‐xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined.The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity.Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe−2S] cluster, and exhibits a typical flavodoxin absorption spectrum.Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected.C component is a Rieske‐type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe−2S] cluster. The cluster is very sensitive to oxygen damage.Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p‐cresol into 4‐methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified.Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.
Immunochemical studies of the lipopolysaccharide O‐specific polysaccharide of Hafnia alvei PCM 1199 related to H. alvei PCM 1205
Authors: Katzenellenbogen Ewa , Kocharova Nina A. , Zatonsky Georgy V. , Mieszala Malgorzata , Gamian Andrzej , Bogulska Maria , Shashkov Aleksander S. , Romanowska Elzbieta , Knirel Yuriy A. 
Published: March 2003

On the basis of chemical analyses and NMR spectroscopic studies, it was found that the O‐specific polysaccharide (O‐PS) isolated from the Hafnia alvei PCM 1199 lipopolysaccharide (LPS) is a glycerol teichoic‐acid‐like polymer having a repeating unit of the following structure :3)‐β‐ D‐Quip4NAc‐(1←1)‐Gro3P‐(O←3)‐β‐
D‐Galp‐(1←3)‐α‐
D‐GlcpNAc‐(1←
2
6
↑∣
1
OAc
β‐
D‐GlcpNAc
6
∣
OAc
where Qui4NAc is 4‐acetamido‐4,6‐dideoxyglucose and O‐acetylation at both positions is non‐stoichiometric. The glycosidic linkage of the lateral β‐
D‐GlcpNAc residue is acid‐labile and cleaved from the OPS during mild acid hydrolysis or dephosphorylation with 48 % hydrofluoric acid. Comparative analysis revealed that the structure of the H. alvei PCM 1199 O‐PS is similar to that of H. alvei PCM 1205, which differs in the presence of an additional lateral α‐
D‐Glcp residue and the position of one of the O‐acetyl groups only. Accordingly, serological tests revealed a high degree of serological similarity between LPSs and O‐PSs of the two H. alvei strains.
Oligosaccharides derived by endo‐β‐galactosidase digestion of bovine corneal keratan sulphate
Authors: Huckerby Thomas N. , Tai Gui‐Hua , Nieduszynski Ian A. 
Published: March 2003

Bovine corneal keratan sulphate has been fragmented using the enzyme endo‐β‐galactosidase and 1H‐NMR chemical shift data are reported for five newly isolated tetrasaccharides which derived from the repeat region. They have the structures : GlcNAc(β1‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal‐ol, GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc(β1‐3)Gal‐ol, GlcNAc(β1‐3)Gal6Sβ(1‐4)GlcNAc6S(β1‐3)Gal‐ol, GlcNAc6S(β1‐3)Gal6S(β1‐4)GlcNAc(β1‐3)Gal‐ol, and GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal‐ol. Structures for two trisaccharides formed by a peeling reaction are also given. These are GlcNAc(β1‐3)Gal6S(β1‐4)GlcNAc‐ol and GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S‐ol where GlcNAc6S‐ol represents N‐acetylglucosaminitol 6‐O‐sulphate.Characterisation of such structures and their spectral assignments will be of considerable value for the studies of both selectin ligands and undersulphated keratan sulphates such as those occurring on cell surfaces and in brain tissue.
Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276
Authors: Gallagher Stephen C. , George Ashley , Dalton Howard 
Published: March 2003

Whole cells of Nocardia corallina B‐276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH‐dependent reductase (reductase) and a regulatory component (coupling protein).The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence‐alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu‐Xaa‐Xaa‐His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active‐site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.
Biosynthesis of the 3‐Acetyl and 131‐Oxo Groups of Bacteriochlorophyll a in the Facultative Aerobic Bacterium, Rhodovulum sulfidophilum
Authors: Porra Robert J. , Urzinger Markus , Winkler Johannes , Bubenzer Claudia , Scheer Hugo 
Published: March 2003

Using 18O‐labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BChl a both aerobically in the dark and anaerobically in the light. R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate 18O label from H218O into both the 131‐oxo and 3‐acetyl oxygens; in addition, the four carboxyl oxygens at C133 and C173 were labelled by H218O. Under aerobic conditions in the dark, the labelling of the 131‐oxo group by H218O was reduced indicating that 16O was being incorporated into this group from air. R. sulfidophilum, grown in the dark under an atmosphere initially containing 50 %18O2 in Ar, possessed an oxygenase which incorporated 18O label from 18O2 specifically into the 131‐oxo group; under these conditions the acetyl and carboxyl groups remained unlabelled.Thus, both an oxygenase and hydratase operate in R. sulfidophilum to form the 131‐oxo group of ring E of BChl a; the 3‐acetyl group oxygen, however, arises only from water via a hydratase.
Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin
Authors: Svensson Stefan , Some Margareta , Lundsjö Anders , Helander Anders , Cronholm Tomas , Höög Jan‐Olov 
Published: March 2003

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class‐I γγ ADH catalyses the dismutation of both acetaldehyde and 5‐hydroxyindole‐3‐acetaldehyde (5‐HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min−1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5‐HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 µm; kcat = 40 min−1). In a system where NAD+ is regenerated, the oxidation of 5‐hydroxytryptophol to 5‐hydroxyindole‐3‐acetic acid proceeds with concentration levels of the intermediary 5‐HIAL expected for a two‐step oxidation. Butanal and 5‐HIAL oxidation is also observed for class‐I ADH in the presence of NADH. The class‐II enzyme is less efficient in aldehyde oxidation, and the ethanol‐oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5‐hydroxyindole‐3‐acetic acid (Ki = 2 mm). Reduction of 5‐HIAL is efficiently catalysed by class‐I γγ ADH (kcat = 400 min−1; Km = 33 µm) in the presence of NADH. This indicates that the increased 5‐hydroxytryptophol/5‐hydroxyindole‐3‐acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class‐I ADH.
A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana
Authors: Faivre‐Nitschke S. Emmanuelle , Grienenberger Jean M. , Gualberto José M. 
Published: March 2003

The gene and cDNA of an Arabidopsis thaliana cytidine deaminase (CDA) were cloned and sequenced. The gene, At‐cda1, is located on chromosome 2 and is expressed in all plant tissues tested, although with quantitative differences. Expression analysis suggest that At‐cda1 probably codes for the housekeeping cytidine deaminase of Arabidopsis. The gene was functionally expressed in Escherichia coli and the protein, At‐CDA1, shows similar enzymatic and substrate specificities as conventional cytidine deaminases: it deaminates cytidine and deoxycytidine and is competitively inhibited by cytosine‐containing compounds. Because the protein shows no affinity to RNA, it is not likely to be involved in RNA‐editing by C‐to‐U deamination. When compared to cytidine deaminases from other organisms, it becomes clear that At‐CDA1 is related, both in sequence and structure, to the CDA of E. coli and other gram‐negative bacteria. The eubacterial nature of the Arabidopsis CDA suggests that it is an additional example of a plant gene of endosymbiotic origin.
Activation of active‐site cysteine residues in the peroxiredoxin‐type tryparedoxin peroxidase of Crithidia fasciculata
Authors: Montemartini Marisa , Kalisz Henryk M. , Hecht Hans‐Jürgen , Steinert Peter , Flohé Leopold 
Published: March 2003

Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide‐detoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. (1997) Biol. Chem.378, 827–836], is a member of the peroxiredoxin family and is characterized by two VCP motifs. Based on a consensus sequence of TXNPx and peroxiredoxin‐type peroxidases, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for α‐helix content by CD and kinetically analysed. The variant Q164E was fully active, C52S, W87D and R128E were inactive and C173S, W87H, W177E and W177H showed reduced activity. Wild‐type TXNPx and Q164E exhibit ping‐pong kinetics with infinite maximum velocities, whereas saturation kinetics were observed with C173S and W177E. The data comply with a mechanism in which C52, primarily activated by R128 and possibly by W87, is first oxidized by hydroperoxide to a sulfenic acid derivative. C173, supported by W177, then forms an intersubunit disulfide bridge with C52. If C173 is exchanged with a redox‐inactive residue (Ser) or is insufficiently activated, the redox shuttle remains restricted to C52. The shift in the kinetic pattern and decrease in specific activity of C173S and W177E may result from a limited accessibility of the oxidized C52 to tryparedoxin, which in the oxidized wild‐type TXNPx presumably attacks the C173 sulfur of the disulfide bridge. The proposed mechanism of action of TXNPx is consistent with that deduced for the homologous thioredoxin peroxidase of yeast [Chae, H. Z., Uhm, T. B. & Rhee, S. G. (1994) Proc. Natl Acad. Sci. USA91, 7022–7026] and is supported by molecular modelling based on the structure of the human peroxiredoxin ‘hORF6’[Choi, H.‐J., Kang, S. W. Yang, C.‐H., Rhee, S. G. & Ryu, S.‐E. (1998) Nat. Struct. Biol.5, 400–406].
Characterization of theRhodobacter sphaeroides5‐aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinantEscherichia coli
Authors: Bolt Edward L. , Kryszak Luiza , Zeilstra‐Ryalls Jill , Shoolingin‐Jordan Peter M. , Warren Martin J. 
Published: March 2003

ThehemAandhemTgenes encoding 5‐aminolaevulinic acid synthase (ALAS) from the photosynthetic bacteriumRhodobacter sphaeroides, were cloned to allow high expression inEscherichia coli. Both HemA and HemT appeared to be activein vivoas plasmids carrying the respective genes complemented anE. coli hemAstrain (glutamyl‐tRNA reductase deficient). The over‐expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activityex vivo. Pure HemA was recovered in yields of 5–7 mg·L−1of starting bacterial culture and pure HemT at 10 mg·L−1. HemA has a final specific activity of 13 U·mg−1with 1 unit defined as 1 µmol of 5‐aminolaevulinic acid formed per hour at 37 °C. TheKmvalues for HemA are 1.9 mmfor glycine and 17 µmfor succinyl‐CoA, with the enzyme showing a turnover number of 430 h−1. In common with other ALASs the recombinantR. sphaeroidesHemA requires pyridoxal 5′‐phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo‐ALAS. Similarly, reduction of the HemA–PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet–visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5′‐phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross‐react with mouse ALAS, indicating that all of the proteins have conserved epitopes.
Cloning, sequencing and heterologous expression of pyrogallol‐phloroglucinol transhydroxylase from Pelobacter acidigallici
Authors: Baas Dietmar , Rétey Jànos 
Published: March 2003

A genomic λ‐library of Pelobacter acidigallici has been established. Proteolytic digestion of homogeneous pyrogallol‐phloroglucinol transhydroxylase from the same microorganism afforded polypeptide fragments whose N‐terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N‐terminal sequences of the two intact subunits these were used in PCR experiments to obtain three amplificates. Screening the λ‐library with the three amplificates led eventually to clones containing the whole gene coding for the transhydroxylase. Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the α‐ and β‐subunits of THL, respectively. The two subunits are separated by a 48‐bp noncoding region. Comparison of the sequence with those of other molybdopterin cofactor (MoCo)‐enzymes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron‐sulphur clusters were spotted. Using the expression vectors pQE 30 and pT 7‐7 three constructs harbouring the THL gene were created. One of them carried a His6‐tag at the N‐terminus of the α‐subunit, another at the C‐terminus of the β‐subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme.
Characterization of recombinant and plant‐derived mistletoe lectin and their B‐chains
Authors: Eck Jürgen , Langer Martin , Möckel Babette , Witthohn Klaus , Zinke Holger , Lentzen Hans 
Published: March 2003

Mistletoe lectin I (pML) and its isoforms ML II and III constitute the active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy. The heterodimeric disulfide‐linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP). Recently, the sequence coding for the mistletoe lectin prepro‐protein was identified and the existence of a single intron‐free gene was shown [Eck, J., Langer, M., Möckel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, H. (1999) Eur. J. Biochem.264, 775–784]. The aim of this study was to prepare pure and homogeneous rMLB‐chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asialofetuin was able to compete with binding of both chains (50% at 0.8 µm). The specificity of the B‐chains to lactose was more distinct with halfmaximal competition at 4.9 mm (pMLB) and > 90 mm (rMLB), respectively. Furthermore, in a coassociation process rMLA‐ and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML‐heterodimer. The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared. Cytotoxicity was determined using MOLT‐4 cells and enzymatic rRNA N‐glycosidase activity was measured in a coupled transcription/translation assay. The IC50 values of the two heterodimers were similar in both assays; rMLB‐chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved at 1.6 mm (rML) and 1.8 mm (pML). Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML. Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activity and the rRNA N‐glycosidase activity, are prerequisites for the cytotoxic effects on target cells.
Lectinochemical characterization of a GalNAc and multi‐Galβ1→4GlcNAc reactive lectin from Wistaria sinensis seeds
Authors: Song Shuh‐chyung , Chen Yuen‐yuen , Herp Anthony , Tsai Ming‐sung , Wu Albert M. 
Published: March 2003

An agglutinin that has high affinity for GalNAcβ1→, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid‐treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 m lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin–glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcβ1→4 containing glycoprotein (GP) [Tamm–Horsfall Sd(a+) GP]; a Galβ1→4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human α1‐acid GP) and a GalNAcα1→3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins.Among the oligosaccharides tested, GalNAcα1→3GalNAcβ1→3Galα1→4Galβ1→4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galβ1→ 3/4GlcNAc (I/II), GalNAcα1→3(l‐Fucα1→2)Gal (Ah), Galβ1→4Glc (L), Galβ1→3GalNAc (T) and Galα1→ 3Galα→methyl (B). Of the monosaccharides and their glycosides tested, p‐nitrophenyl βGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding α anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal.From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C‐3, C‐4 and C‐6 positions of GalNAc and Gal residues; the N‐acetyl group at C‐2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both α and β anomers of GalNAc. Gal ligands must be Galβ1→3/4GlcNAc related, in which subterminal β1→3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the β anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp ≥ multi‐II > monomeric II ≥ Tn, I and Ah ≥ E and L > T > Gal.