bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct refinement of the atomic structure of MoS2layered compounds from PXRD
Authors: Bushmarinov Ivan , Goloveshkin Alexander , Golub Alexandre
Published: November 2015

The graphene analog MoS2can be exfoliated into single-layer dispersion of MoS2crystals by reduction of starting material to LiMoS2followed by detachment of its layers in aqueous solvents. This dispersion reacts with organic salts in solution, producing precipitates of layered MoS2intercalated with their cations [1]. Most of these compounds have distinct chemical composition and powder X-ray diffraction (PXRD) patterns. The analysis of published data, however, indicates that these diffraction patterns were used only for fingerprinting and estimation of cation size and orientation by checking the positions of the basal (00l) reflections. The low-intensity hkl zone of the diffraction patterns was effectively ignored. In the current work, we report that the diffraction patterns of MoS2intercalation compounds contain enough information to refine their atomic structure directly. We found that the patterns of (NR4)x MoS2(R=alkyl, H) compounds correspond to turbostratic disorder of MoS2-organic layers. The patterns were indexed with a common orthorhombic cell. The cell content was Rietveld refined using a modification of a "supercell approach" [2,3] developed for full-pattern modeling of disordered clays. We determined that the Mo atoms in MoS2layers in the intercalation compounds change the coordination from prismatic to octahedral and that the S atoms form "nanorunnels" containing organic cations. The geometry of the Mo-Mo chains forming in the layer was consistent with the EXAFS data. The ordering of the organic layer depended on the nature of the cation, with (NEt4)x MoS2notably ordered enough to have specific stoichiometric composition (x=1/6) defined by the intralayer packing. The PW-DFT-d calculations based on our model confirmed our results. The developed approach allows determination of previously inaccessible atomic structures of a wide class of MoS2-based layered compounds using commercially available software and laboratory X-ray diffraction data.
Mode Analysis of the High Temperature Behaviour of some Ferroic Perovskites
Authors: Lightfoot Philip
Published: November 2015

The advent of user-friendly on-line software (such as ISODISTORT [1] and AMPLIMODES [2]) to analyse crystal structures in terms of `distortion modes' rather than the conventional approach of refining individual atomic coordinates, has opened up new possibilities in the understanding of structure-property relationships in functional materials. The perovskite family is ideally suited to this approach, with many different variants on the basic cubic structure-type being brought about by simple symmetry-lowering modes such as tilting or distortion of almost rigid metal-ligand octahedral groups. In this talk I will illustrate the use of distortion mode analysis in understanding the sometimes complex, unusual and surprising structural behavior of several functional perovskites, particularly ferroelectrics and multiferroics. Examples chosen will exploit the use of powder neutron diffraction as the primary experimental technique, and will include sodium niobate, NaNbO3, which has the most complex high-temperature phase diagram of any simple perovskite, and derivatives of BiFeO3 (such as La or Mn-doped) which display unusual magnetostrictive behaviour.
In situ growth of SrFe12O19
Authors: Granados Cecilia , Bøjesen Espen , Jensen Kirsten , Christensen Mogens
Published: November 2015

SrFe12O19 is a highly anisotropic ferrimagnetic compound with relatively high remanence and high coercivity, which is used in permanent magnets. Permanent magnets are everywhere in our daily life and they are responsible for the interconversion between motion and electricity in electrical components ranging from headphones to wind turbines. Three key parameters, important for making permanent magnets, are an anisotropic structure, size of the nanocrystallites and the microstructure. In situ X-ray powder diffraction has been used to follow the growth kinetics of SrFe12O19 under hydrothermal conditions. Synthesis of SrFe12O19 (Sr-Hexaferrite) nanocrystals by hydrothermal methods have the advantage of allowing exhaustive control of the reaction parameters. We have studied the growth and kinetics of SrFe12O19 by carring out time resolved synchrotron experiments at MAX-lab, Sweden. The experiments were carried out at elevated pressure (250 bar) and in temperatures ranging from 250 to 400 oC. The diffraction data allow us to follow the evolution of the crystallite size as function of temperature, time and composition. By controlling the composition of the precursor we can tailor the size of the nanocrystallites. The obtained data have shown that the synthesis takes place through a conversion of tiny hexagonal shaped FeOOH nanocrystallites into the SrFe12O19. Several ex situ studies under comparable conditions have been carried out to compare the magnetic properties and the obtained nanocrystallites have been investigated using high resolution laboratory powder diffraction data.
Thermoresponsive studies of porous and non-porous borophosphates.
Authors: Mogodi Mashikoane , Billing David
Published: November 2015

In this work we present the synthesis and structure-property investigation of anhydrous low porosity borophsophates [1] BPO4 and porous (NH4)0.5M1.25(H2O)2)(BP2O8)(H2O)0.5 M = Co (II), Mn (II) and NH4Fe[BP2O8(OH)] phases. Cristobalite-type BPO4 crystallizes in the tetragonal lattice, space group I-4 (No. 82) [2]. Variable-Temperature Powder X-ray diffraction (VT-PXRD) patterns for these material were analysed by the sequential and parametric Rietveld refinement [3] protocols. Both methods were used to determine the temperature dependency of the lattice parameters and linear thermal expansion coefficient. Whereas the lattice parameters were refined freely in the sequential method, the individual cell parameters were described using an empirically derived function in the parametric method. Both refinement protocols reveal significant anisotropy along the a- and c- axis as a function of temperature, with thermal expansion coefficients of 10.6 x10-6 /0C and 2.83 x10-6 /0C, respectively. Structural changes accompanying this thermoresponsive behaviour will be discussed, including the variation of the interatomic distances and P-O-B (inter-polyhedral angle, figure 1) with temperature. The open framework (NH4)0.5M1.25(H2O)2)(BP2O8)(H2O)0.5 M = Co(II) (1a), Mn(II) (1b) and NH4Fe[BP2O8(OH)] (2) phases were synthesized under mild hydrothermal methods at 1800C. The crystal structure of the isostructural (1a) and (1b) phases were refined in the hexagonal lattice, space group P65 (no. 170) and compound (2) in the monoclinic lattice, space group P21/c (no.14). Both (1a) and (1b) phases consists of NH4+ and H2O molecules located within the helical channels running along the [001] direction with compound (2) consisting exclusively of NH4+ molecules located within the helical channels running along the [100] direction. Thermoresponsive investigation conducted by TGA analysis reveal a five, four and three step mass loss process for compounds (1a), (1b) and (2) respectively, with the final step observed at 500-7000C range. Preliminary VT-PXRD results of these compounds will also be presented.
A closed-homodimer structure of human importin-α1
Authors: Miyatake Hideyuki , Sanjoh Akira , Matsuda Go , Tatsumi Yuko , Miyamoto Yoichi , Dohmae Naoshi , Aida Yoko
Published: November 2015

We determined the crystal structure of N-terminal importin-β-binding (IBB) domain truncated human importin-α1 (ΔIBB-importin-α1) at 2.63Å resolution. The crystal structure of ΔIBB-importin-α1 is a novel closed-homodimer. The homodimer exists in an autoinhibition state in which both of the major and minor NLS-binding sites are completely buried in the homodimerization interface to avoid NLS binding. Importin-α1 is in dimer-monomer equilibration in solution. In the dimerization sate, the P1'-binding pocket in the minor NLS binding site plays a role to stabilize the dimer formation. The external K108 binds into the P1'-binding pocket that results in the autoinhibition of the NLS binding. The present closed-homodimer of ΔIBB-importin-α1 conjured the functional aspects of multimerization of importin-α1. The further physicochemical studies using full- and ΔIBB- importin-α1 reveal that the IBB domain is involved in the monomer-dimer equilibration; thereby the NLS binding affinity is kept even in the higher concentration of importin-α1. Owing to the multimerization property, importin-αs can autoinhibition the NLS binding, that may result in a variety of NLS recognition way.
Conformation-independent structural comparison of macromolecules with ProSMART
Authors: Nicholls Robert , Fischer Marcus , Murshudov Garib
Published: November 2015

Structural comparison often aids insight into the function and mechanics of biological macromolecules. To make such analyses more accessible, we present the Procrustes Structural Matching Alignment and Restraints Tool (ProSMART), which is designed to allow fast but detailed comparative analysis of macromolecular structures despite potential dissimilarities in global arrangement, such as domain motion and distortion. Whilst obtaining a residue alignment between structures is a prerequisite for comparative analysis, conventional alignment methods may fail in cases where conformational differences are dramatic. However, ProSMART achieves a conformation-independent structural alignment by focusing purely on local dissimilarities, rather than enforcing chain/domain rigidity. This allows the sensible comparison of protein (or DNA/RNA) structures in the presence of conformational change. ProSMART allows analysis of the structural conservation of local backbone and side chains in a wide variety of scenarios - the method is sensitive enough to allow identification of subtle dissimilarities between structures sharing high sequence homology, whilst being versatile enough to allow identification of local similarities between more distantly-related structures. In addition, ProSMART can be used for the identification of conserved rigid substructures, which may or may not represent functional domains. ProSMART is also used for the generation of external restraints for use in crystallographic refinement. Results from ProSMART can be visualised in either CCP4mg or PyMOL. All residue-based scores are illustrated using intuitive colour gradients, allowing easy visual assessment of local backbone and side chain conservation. Complementary structural comparison tools such as ProSMART can help break the complexity of the constantly growing pool of available structural data into a more readily accessible form, and consequently may aid biological insight into macromolecular structures.
Expanding our knowledge of the protein universe: Modelling of protein structures
Authors: Haas Jürgen , Barbato Alessandro , Schmidt Tobias , Roth Steven , Waterhouse Andrew , Bienert Stefan , Arnold Konstantin , Bordoli Lorenza , Schwede Torsten
Published: November 2015

Computational modeling and prediction of three-dimensional macromolecular structures and complexes from their sequence has been a long standing goal in structural biology. Over the last two decades, a paradigm shift has occurred: starting from a large "knowledge gap" between the huge number of protein sequences compared to a small number of experimentally known structures, today, some form of structural information – either experimental or computational – is available for the majority of amino acids encoded by common model organism genomes. Methods for structure modeling and prediction have made substantial progress of the last decades, and template based homology modeling techniques have matured to a point where they are now routinely used to complement experimental techniques. However, computational modeling and prediction techniques often fall short in accuracy compared to high-resolution experimental structures, and it is often difficult to convey the expected accuracy and structural variability of a specific model. Retrospectively assessing the quality of blind structure prediction in comparison to experimental reference structures allows benchmarking the state-of-the-art in structure prediction and identifying areas which need further development. The Critical Assessment of Structure Prediction (CASP) experiment has for the last 20 years assessed the progress in the field of protein structure modeling based on predictions for ca. 100 blind prediction targets per experiment which are carefully evaluated by human experts. The "Continuous Model EvaluatiOn" (CAMEO) project aims to provide a fully automated blind assessment for prediction servers based on weekly pre-released sequences of the Protein Data Bank PDB. CAMEO has been made possible by the development of novel scoring methods such as lDDT, which are robust against domain movements to allow for automated continuous structure comparison without human intervention.
Kinetic Crystallography on MutT and its homolog
Authors: Nakamura Teruya , Hirata Keisuke , Yoshikawa Kohei , Inazato Miyuki , Chirifu Mami , SIkemizu Shinji , Yamagata Yuriko
Published: November 2015

Oxidized deoxynucleotides cause replicational errors because of their misincorporations into DNA. The MutT and related proteins prevent transversion mutations by hydrolyzing mutagenic oxidized nucleotides such as 8-oxo-dGTP and 2-oxo-dATP, and there is a difference in substrate specificities between them. E. coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP with extremely high substrate specificity. On the other hand, its human homolog has broad substrate specificity for oxidized nucleotides and hydrolyzes 8-oxo-dGTP as well as 2-oxo-dATP. In order to understand mechanisms of their substrate specificities, we solved the crystal structures of MutT and its homolog complexed with their substrates and revealed structural basis of the high substrate specificity of E. coli MutT for 8-oxoguanine nucleotide and the broad substrate specificity of its human honolog for oxidized nucleotides. In this paper, we report the hydrolysis mechanisms of both enzymes revealed by kinetic protein crystallography. Both hydrolysis reactions were initiated by soaking the enzyme-substrate complex crystals in divalent metal solution. After incubation under various conditions, the reactions were terminated by freezing the crystals at 100K. X-ray diffraction data were collected at Spring-8 and Photon Factory. In the MutT crystals, the structures of sequential catalytic intermediates showed the activation mechanism of the nucleophilic water molecule synchronized with the coordination of metal ions. Now by using the crystals of its human homolog, the trial of the catching the intermediate state of catalysis is in progress.
New Insights on Structure and Function of Sialyltransferases
Authors: Volkers Gesa , Worrall Liam , Lameignere Emilie , Strynadka Natalie
Published: November 2015

Sialic acids are a unique posttranslational modification at the terminus of glycoproteins and -lipids. Proteins modified with oligomers of sialic acid add a repellent charge to cell surfaces, which is a crucial feature in cell migration and axonal growth during early brain development. Varied expression levels of sialic acid are linked to tumor malignancy in neuroblastoma, schizophrenia, autism and bipolar disorder but the lack thereof is linked to impaired neuronal development. On the other hand, overexpression of sialic acid oligomers in Schwann cells promotes the peripheral regeneration of lesioned nerves and improves the ability of Schwann cells to migrate into damaged tissue and to remyelinate central nervous system axons. In order to understand the molecular mechanisms of sialylation, our project focuses on the structural characterization of enzymes of the mammalian and bacterial glycosyltransferase families 29 and 42. The proteins of interest were expressed in insect cells and structural studies were undertaken by x-ray crystallography. Kinetics, SEC MALS and glycan array data will shed light on mechanism of catalysis and acceptor specificity. Altogether, the results of this study will promote further understanding of the structure-function relationship of sialyltransferases.
Structural characterization of heparinase III from Bacteroides thetaiotaomicron
Authors: Ulaganathan Thirumalaiselvi , Cherney Maia , Yao Deqiang , Li Guoyun , Li Lingyun , Linhardt Robert , Cygler Miroslaw
Published: November 2015

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid (GlcA) or iduronic acid (IdoA), linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc). GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix (ECM), control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior, and differentiation. Niche adapted bacteria expresses enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. Polysaccharide lyases have been classified into 23 sequence-related families. Comparison of three-dimensional structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level and identified occurrences of convergent evolution. We have characterized structurally and enzymatically Heparinase III (HepIII) from Bacteroides thetaiotaomicron, which is classified within the PL12 family. HepIII is a 72.5KDa protein. We will present the X-Ray structures of two crystal forms of HepIII of resolution 1.8 Å and 2.6 Å. HepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal β-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of HepIII toward sulfate poor heparan sulcate substrate.
Structural Studies of an Novel Sugar Nucleotide Pyranose-Furanose Mutase
Authors: Sanders David , Dalrymple Sean , Poulin Myles , Zheng Ruixiang , Lowary Todd
Published: November 2015

Campylobacter jejuni (Cjj) is the leading cause of gastroenteritis in humans, its glycoconjugates are known to include sugars in the furanose ring conformation. In particular, Cjj HS:41 strains are associated with the development of Guillian-Barré syndrome (GBS) and produce a key virulence factor of the bacterium, namely furanose-based capsular polysaccharides (CPS). To date, the enzymes responsible for furanose biosynthesis in Cjj HS:41 CPS are poorly understood. CPS sequencing has revealed three genes, annotated as glf1-3 via homology to known bacterial UDP-D-galactopyranose mutase (UGM), could be involved in the biosynthetic pathways. Our laboratories are interested in the structure-function relationship behind recognition and discrimination within such pyranose-furanose mutases. Enzymology studies have shown Glf1 is a flavoprotein responsible for isomerization between GDP-6d-D-altro-heptopyranose and GDP-6d-D-altro-heptofuranose, the latter being a major component of CPS. This GDP-altro-heptopyranose mutase (GaHM) activity is the first example of a heptose-recognizing mutase. Given that many of the UGM active site and FAD binding site residues are conserved in GaHM, the catalytic mechanism is likely similar to that of UGM. In order to establish key features of the enzyme, detailed structural information is required. We report here on the structure of Cjj GaHM, which has been co-crystallized with GDP, in both oxidized and reduced states. Due to low sequence identity with bacterial UGMs, Se-MET SAD phasing was ultimately employed to solve the structure. We will also discuss our recent crystallization efforts with Cjj GaHM in the presence of GDP-sugar substrate derivatives. Ultimately, the structural information gleaned from this study could lead to the identification of a new inhibitors targeting the CPS biosynthetic pathway.
Partial oxygen-dissociation of crystalline giant hemoglobin
Authors: Numoto Nobutaka , Nakagawa Taro , Kita Akiko , Ito Nobutoshi , Fukumori Yoshihiro , Miki Kunio
Published: November 2015

Allosteric oxygen-binding of hemoglobin (Hb) has been widely discussed based on the quaternary structural changes elucidated by the crystal structures of the oxygenated and deoxygenated states. However, it remains to be determined the structure of intermediate states between the oxy and deoxy forms without any artificial modification of the Hb molecule. A tubeworm, Lamellibrachia satsuma has extracellular giant hemoglobins with a molecular mass of about 400 and 3,600 kDa. Recently, we have determined the crystal structure of the 400 kDa Hb (V2Hb) in the oxy state, and then we successfully obtained the deoxygenated crystals of V2Hb from oxy crystals by the soaking methods [1]. These findings encourage us to initiate structural studies for the intermediate states between the oxy and deoxy forms of V2Hb, which should provide a more accurate understanding of the allosteric mechanism of Hbs. The deoxy crystals of V2Hb were obtained from oxy crystals through the soaking in a solution containing 50 mM sodium hydrosulfite, and incubated for a few minutes. We tested various soaking times from 3 s to 180 s and then immediately flash-frozen under a nitrogen gas stream. The obtained structures reveal that in the case of the soaking time was longer than 10 s, the electron densities of the oxygen molecules at some heme pockets (oxygen binding sites) were very week or disappeared. These `intermediate' structures show almost the same quaternary structure as that of the oxy structure. This fact suggests that quaternary rearrangement of V2Hb might arise just before a complete dissociation of all the oxygen molecules from all the subunits.
Structural characterization of Polycyclic Aromatic Hydrocarbon degrading enzymes
Authors: Coitinho Juliana , Costa Debora , Guimaraes Samuel , Neves Cintia , Araujo Simara , Costa Mariana , Nagem Ronaldo
Published: November 2015

Polycyclic aromatic hydrocarbons (PAHs) are a group of aromatic hydrocarbons composed of two or more fused rings and include naphthalene, phenanthrene, pyrene and benzo[a]pyrene amongst others. Exposure to naphthalene has been associated with several toxic manifestations in humans and laboratory animals, with the lens of the eye and the lungs being most sensitive [1]. Additionally, this compound has been reclassified as a possible human carcinogen by the US Environmental Protection Agency and the International Agency for Research on Cancer. It has recently been suggested that naphthalene undergoes metabolic activation to 1,2-naphthoquinone, which reacts with DNA, leading to the formation of apurinic sites on DNA and depurinating DNA adducts [2]. Moreover, a number of studies have attested to the carcinogenic and mutagenic properties of more complex PAHs, suggesting the need for further work on the elimination of these compounds from the environment. The Gram-negative bacterium P. putida G7 is among the best studied naphthalene-degrading species. The genes associated with naphthalene metabolism are localized on NAH7, an 83 kilobase plasmid. In P. putida G7 the naphthalene-oxidation genes are organized into two operons under salicylate control. The first operon (upper naphthalene-degradation pathway) includes the genes nahAaAbAcAdBCDEF, which code for the conversion of naphthalene to salicylate, while the second operon (lower pathway) includes the genes nahGTHINLOMKJ responsible for the oxidation of salicylate via the catechol meta-cleavage pathway [3]. In the last years, our group focused on the structure elucidation and the kinetic characterization of the P. putida G7 enzymes involved in the naphthalene degradation pathway. We intend to present these results which basically describe the 3D structures of NahB, NahF, NahG, NahI, NahK and NahK/NahL complex and some kinetic data of these enzymes and their mutants. This work was supported by FAPEMIG, CNPq and VALE S.A.
Structure and Function Analysis of Geobacillus Starch Antistaling Enzyme
Authors: Mikami Bunzo , Bitoh Hirotaka , Anzai Yurie , Mizutani Kimihiko , Takahashi Nobuyuki , Okada Masamichi , Yamaguchi Shotaro
Published: November 2015

Glycosyltransferase from Geobacillus sp. (SAS) is expected to see wide use as a starch antistaling enzyme in food including bread and rice products. The enzyme is thought to transfer maltotriose (G3) unit into non-reducing ends of sarch with unknown likage except for usual alpha-1,4 linkage. SAS was crystallized by sitting drop vapar diffusion method in 14~28% PEG4000 (w/v), 10mM CaCl2, 0.1M NaAC at pH 4.6 and 20°C for 1 month. The obtained crystals belong to a space group of P6522 with cell dimensions of a = b = 112 and c = 320 Å. The crystals were soaked in various oligomaltosaccharides (G1, G2, G3, G4, G5 and G6) for 15 min before flash cooling. The diffraction data of each complex were collected at beam-lines of BL26B1, BL38B1 and BL44XU in SPring-8. The crystal data were collected with 97-99 % completeness and Rmerge of 0.07-0.09 up to 1.6-2.3 Å resolution. The structures were determined by molecular replacement with cyclodextrin glucanotransferase (CGTase, PDB 1CYG) as a search model and were refined with PHENIX. The refined models of SAS/sugars contain one molecule of SAS comprising 733 amino acid residues, 5-8 calcium ions, 543-1141 water molecules and several sugars with R = 0.15-0.19 and Rfree = 0.16-0.23 for the data up to 1.6-2.3 Å resolution. SAS has almost the same overall structure with the CGTase except for several loops in the catalytic domain A. They share a similar active site except for subsite +3 where the non-reducing ends of the oligosaccharides bind. G1 bound to subsite +3, indicating +3 site has the highest affinity to G1. Only G3 was found to bind at subsites +3 ~ +1 when G3, G5 and G6 were soaked, whereas G4 bound at subsites +3 ~ -1 when G4 was soaked. From the clear density map of the bound G4, the bound glucose residue at subsute -1 is found to have alpha-1,6 linkage, indicating the product of this transglucosidase.
De-N-acetylation and Export of the Biofilm Exopolysaccharide PNAG
Authors: Little Dustin , Li Grace , Ing Christopher , Poloczek Joanna , DiFrancesco Benjamin , Bamford Natalie , Robinson Howard , Nitz Mark , Pomès Régis , Howell P
Published: November 2015

Bacteria embedded in a self-produced matrix of exopolymeric substances, or biofilm, represent a significant medical problem, as the bacteria are tolerant to antibiotics, protected from the environment, and isolated from the innate immune system. A key component required for the development of the biofilm in a wide variety of pathogenic bacteria is the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG). Four proteins, PgaA/B/C/D, are required for the polymerization, modification, and export of PNAG in Escherichia coli. PgaB is a two-domain outer membrane lipoprotein essential for the partial de-N-acetylation of PNAG (dPNAG); a process required for polymer export and subsequent biofilm formation. Here we report 1.9 Å and 1.8 Å crystal structures of PgaB and its isolated C-terminal domain in complex with a PNAG hexamer, respectively. Characterization of PgaB de-N-acetylase activity using PNAG oligomers reveals that the enzyme has low catalytic efficiency, and displays length- and metal-dependent de-N-acetylation activity specific for PNAG with preference for Fe2+, Ni2+, and Co2+. Furthermore, tryptophan fluorescence quenching assays show the C-terminal domain binds PNAG oligomers with ~1-4 mM affinity. These data in combination with molecular dynamics simulations of PgaB with N-acetylglucosamine and glucosamine suggest PNAG de-N-acetylation occurs first, with subsequent binding of dPNAG to the C-terminal domain. We propose this concerted action plays a pivotal role in targeting dPNAG for export through the outer membrane porin PgaA.
Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa
Authors: Lee Michael , Schep Daniel , McLaughlin Brian , Kaufmann Martin , Jia Zongchao
Published: November 2015

Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts, and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 1800across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor, and produces five times more CO than ChuS. Addition of catalase slows down, but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.
Structural analysis of E. coli 6-carboxytetrahydropterin synthase
Authors: Killivalavan Asaithambi , Seo Kyung , Zhuang Ningning , Park Young , Lee Kon
Published: November 2015

The Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homolog of 6-pyruvoyl tetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro rather than the genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. We have determined crystal structures of a wild type apo-eCTPS and a Cys27Ala mutant eCTPS complexed with sepiapterin up to 2.3 and 2.5 Å, respectively. The structures are highly conserved at the active site and the Zn2+ binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologs revealed that two specific residues Trp51 and Phe55, not existing in the mammalian PTPS, kept the substrate bound by stacking it with their side chains. Replacements of these two residues by site-directed mutagenesis to the residues, Met and Leu, existing only in mammalian PTPS, converted the eCTPS to have the mammalian PTPS activity. Our studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and for the specific enzyme activity different from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologs.
Crystal structure of amylomaltase from Corynebacterium glutamicum
Authors: Krusong Kuakarun , Srisimarat Wiraya , Pongsawasdi Piamsook
Published: November 2015

Amylomaltase (AM; EC 2.4.1.25) belongs to the 4-α-glucanotransferase (4αGTase) group of the α-amylase family. The enzyme can produce cycloamylose (CA) or large-ring cyclodextrin (LR-CD) through intramolecular transglycosylation or cyclization reactions of α-1,4 glucan. Amylomaltase from the mesophilic bacterium Corynebacterium glutamicum yielded different LR-CD production profile from that of the well-characterized Thermus aquaticus enzyme [1,2]. C. glutamicum amylomaltase (CgAM) was overexpressed, purified and crystallized [3]. X-ray crystal structure of CgAM differs from Th. aquaticus amylomaltase in the presence of an additional N-terminus domain. The acarbose- and maltotriose- bound structures revealed the residues involved in substrate binding.
Crystallographic and kinetic study on riboflavin synthase from Brucella abortus
Authors: Serer María , Bonomi Hernán , Guimarães Beatriz , Rossi Rolando , Goldbaum Fernando , Klinke Sebastián
Published: November 2015

Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione [1]. Due to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3-symmetry [2]. Every monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighboring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the etiological agent of the disease brucellosis) in its apo form, in complex with riboflavin, and in complex with two different product analogues, being the first time in which an intact RS trimer is solved with bound ligands. These crystal models support the hypothesis for an enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.
Structure of hydrogen sulfide-producing enzyme from a periodontal pathogen
Authors: Kezuka Yuichiro , Yoshida Yasuo , Nonaka Takamasa
Published: November 2015

Hydrogen sulfide (H2S) is one of the predominant volatile sulfur compounds that are primarily responsible for oral malodor and contribute to the progress of periodontal disease. H2S in the human oral cavity is generally produced by enzymatic actions of oral bacteria.Fusobacterium nucleatum, a Gram negative periodontal pathogen, is known to be one of the heaviest H2S producers [1]. For now, four genes (fn0625,fn1055,fn1220, andfn1419) encoding pyridoxal-5′-phosphate (PLP)-dependent H2S-producing enzymes have been identified and characterized inF. nucleatumATCC 25586. Of the four enzymes, Fn1055 protein is a unique H2S-producing enzyme, which produces H2S and L-serine from L-cysteine [2]. Therefore, Fn1055 might play important roles in L-serine biosynthesis in addition to H2S production in this periodontal pathogen. Crystal structures of recombinant Fn1055 and its site-directed mutant complex with L-cysteine (a substrate) were determined at 2.1 Å resolution. The enzyme forms a homodimer whose subunits are related by a two-fold axis. The subunit is composed of two domains with α/β structure. The PLP cofactor forms a covalent internal aldimine linkage with the ε-amino group of Lys46 at the bottom of active site cleft between the domains, in the absence of substrate. On the other hand, in the cocrystal of mutant with L-cysteine, the introduced L-cysteine was found to be covalently bound to PLP, instead of Lys46. This covalent intermediate was identified as an α-aminoacrylate, which is the key species of PLP-dependent-enzyme catalysis, by spectrophotometric measurement. Along with the intermediate formation, closure of active site cleft was also observed. Furthermore, we found an amino acid residue acting as a base and confirmed its indispensability for catalysis by enzymatic analyses. These results support that H2S production by Fn1055 proceeds through the β-elimination of L-cysteine, and enable us to propose a detailed catalytic mechanism of Fn1055.
Shikimate kinase-like 1 plays an integral role in chloroplast biogenesis
Authors: Milot Marie-Michelle , Hofmann Adrian
Published: November 2015

The shikimate pathway is found in plants, fungi, and microbes, but not mammals which makes it an attractive target for antimicrobials and herbicide development. In plants, the shikimate pathway produces precursors of compounds that are crucial for growth, defense and, survival. We focused on the enzyme shikimate kinase (SK) which catalyzes the phosphorylation of shikimate; the fifth reaction of the pathway. Plant SK underwent a gene duplication event yielding shikimate kinase-like genes (SKL1 and SKL2) roughly 400 MYA. Despite the overall similarity in sequence between SK and SKL1, a previous in vitro analysis showed that SKL1 does not catalyze a phosphorylation reaction in the presence of shikimate. When a three-dimensional structural model of SKL1 was compared with the microbial crystal structure of SK, we identified several highly conserved functional domains crucial for the shikimate kinase reaction: the shikimate binding domain (SBD) critical for substrate recognition, the LID domain integral for the transfer of phosphate to bound shikimate, and the walker A and B domains important for ATP binding. Of these domains, only the walker A and B domains are conserved in SKL1. Together with in vitro analysis, it is evident that SKL1 does not interact with shikimate, but the function is still unknown. Sequence alignment and modeled structure also revealed the presence of a novel phosphoglycerate mutase-like (PGML) domain within SKL1.This further suggests neofunctionalization of SKL1 compared to its parent gene SK. Current studies in our lab focus on determining the crystal structure of SKL1. This structure will be used for ligand docking experiments to identify potential substrates of the enzyme. As an additional means of substrate identification, we will also be performing crystal soaking experiments using plant extracts.
A special halide-stabilizing pair and enantioselectivity mechanism of DatA
Authors: Guan Lijun , Yabuki Hideya , Okai Masahiko , Ohtsuka Jun , Tanokura Masaru
Published: November 2015

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. DatA possesses a unique Asn-Tyr residue pair instead of the Asn-Trp residue pair conserved among the subfamily members, thus the structural basis for its reaction mechanism merits elucidation. In addition, DatA is potentially useful for pharmaceutical and environmental applications, though several crystal structures of HLD-II dehalogenases have been reported so far, the determination of the DatA structure will provide an important contribution to those fields. This work provided insight into the reaction mechanism of DatA via a combination of X-ray crystallographic and computational analysis. The crystal structures of DatA and the Y109W mutant were determined at 1.70 Å [1] and 1.95 Å, respectively. The location of the active site was confirmed by using its novel competitive inhibitor, CHES. The structural information from these two crystal structures and the docking simulation with 1,3-dibromopropane revealed that the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for 1,3-dibromopropane, due to the extra hydrogen bond between Trp109 and halogenated compounds; and that the key residue to bind halogenated substrate is only Asn43 in the wild-type DatA, while those in the Y109W mutant are the Asn-Trp pair. Furthermore, docking simulation using the crystal structures of DatA and some chiral compounds indicated that enantioselectivity of DatA toward brominated alkanes is determined by the large and small spaces around the halogen binding site.
Crystal structure analyses of oxygenase component of resorcinol hydroxylase
Authors: Fujii Tomomi , Kobayashi Kazutaka , Yamauchi Takae , Yoshida Masahiro , Oikawa Tadao , Hata Yasuo
Published: November 2015

The resorcinol hydroxylase is involved in the first step of the resorcinol catabolic pathway and catalyzes hydroxylation of resorcinol to hydroxyquinol. The enzyme belongs to the two-component flavin-diffusible monooxygenase family and acts in the coexistence of two components: an oxygenase and a flavin reductase. The oxygenase component hydroxylates the substrate using molecular oxygen and reduced flavin produced by the reductase. To understand the structural basis for the catalytic mechanism, we analyzed the crystal structure of the oxygenase component (GraA) from Rhizobium sp. strain MTP-10005. The GraA subunit has 409 amino acid residues. Apo-form crystals were obtained in the tetragonal space group I4122 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and K2HPO4. Holo-form crystals were obtained in the trigonal space group P3221 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and KNO3. Both structures were determined by molecular replacement and refined at 2.3 Å and 3.2 Å resolutions, respectively. GraA is a homotetramer with three molecular two-fold axes identical to crystallographic two-fold axes in the apo-form crystal. In the holo-form crystal, four tetramers exist in the asymmetric unit and each subunit binds one FAD. The subunit consists of three domains. The N-terminal domain has an α-structure mainly of antiparallel α-helices; the central domain has a β-structure of two β-sheets stacked together; the C-terminal domain has a four-helix-bundle structure of long antiparallel α-helices involved in tetramer formation. In the holo-form, the FAD is located in the space that is encompassed by these three domains. The loop region of 13 residues, which is disordered in the apo-form, is ordered and covers FAD of another subunit. The turn portion of the loop occludes the entrance of the putative active site.
Crystallization and preliminary X-ray diffraction analysis of nepenthesin-1
Authors: Fejfarová Karla , Man Petr , Mrázek Hynek , Kádek Alan , Halada Petr , Skálová Tereza , Kolenko Petr , Švecová Leona , Hašek Jindřich , Dohnálek Jan
Published: November 2015

Carnivorous pitcher plants of the genus Nepenthes secrete their own aspartic proteases, nepenthesins, to digest prey. Nepenthesins differ significantly in sequence from other plant aspartic proteases. This difference, which brings more cysteine residues into the structure of nepenthesins, in conjunction with putative N-glycosylation, can contribute to uniquely high temperature and pH stabilities of these proteases [1, 2]. In continuation of our previous study of the expression and biochemical and enzymatic characterization of a recombinant form of nepenthesin-1 (rNep-1) from Nepenthes gracilis [3], we report its crystallization and preliminary X-ray analysis. Crystals of rNep-1 in complex with the pepstatin A inhibitor have been grown using the hanging-drop vapour-diffusion technique. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at Bessy II of HZB, Berlin. The crystals belong to space group P21, with unit-cell parameters a = 54.4 Å, b = 86.6 Å, c = 95.8 Å, β = 1060. The self-rotation function combined with solvent-content calculations and Matthews coefficient suggest the presence of two molecules of rNep-1 in the asymmetric unit. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grants No. EE2.3.30.0029 and No. LG14009), by BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund, and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14.
Structural insights into broad substrate range of biphenyl dioxygenases & dehydrogenase
Authors: Dhindwal Sonali , Sylvestre Michel , Kumar Pravindra
Published: November 2015

Biphenyl dioxygenase(BphAE) & biphenyl dehydrogenase(BphB) catalyze first two steps of aerobic degradation of biphenyl & various polychlorinated biphenyls,PCBs. These enzymes are of interest for their potential to oxidize toxic pollutants & manufacture of fine chemicals. We have determined the crystal structures of two variants of BphAE from B. xenovorans LB400, viz BphAERR41 & BphAEp4, evolved by site directed mutagenesis. We have compared the structures of wild type BphAELB400 & BphAEp4. Biochemical properties of BphAELB400 variants with single substitutions, T335A or F336M shows that residue 336 contacts biphenyl & influences regiospecificity of the reaction, but do not enhance enzyme's reactivity toward 2,6-dichlorobiphenyl. However, residue 335 does not contact biphenyl but contributes significantly to expand enzyme's substrate range. Crystal structure of variant BphAERR41 explains the transformation of dibenzofuran & 2-chlorodibenzofuran. This study provides structural insight for expanded substrate range of Rieske-type oxygenases through mutations that enhance the plasticity and/or mobility of enzyme segments lining the catalytic site. Enzyme, BphB catalyze 2nd step of PCB degradation pathway. We have determined the crystal structure of apoenzyme, binary complex with NAD+ & ternary complexes with NAD+2,3-dihydroxybiphenyl from P. pnomenusa B356. A crystal structure representing an intermediate state of the enzyme was also obtained where the substrate binding loop was ordered in comparison with apo & binary forms but was displaced significantly w.r.t ternary structure. These structures reveal that the substrate binding loop is highly mobile & changes conformation during ligand binding, starting from a disorganized loop in apo state to a well organized loop in ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate wide range of substrates. For the 1st time, a combination of structural, biochemical, & molecular docking studies of BphBB356 elucidate the unique ability of enzyme to transform the cis-dihydrodiols of double meta-, para-, & ortho-substituted chlorobiphenyls.
Allosteric Signaling in Dihydrodipicolinate Synthase
Authors: Conly Cuylar , Skovpen Yulia , Palmer David , Sanders David
Published: November 2015

Dihydrodipicolinate synthase (DHDPS) is an enzyme found in most bacterial species and regulates the production of cell wall precursors necessary for the life of the organism. Specifically, DHDPS catalyzes the condensation of pyruvate and aspartate-β-semialdehyde (ASA) to produce dihydrodipicolinic acid leading to the synthesis of cell wall precursors lysine and mesodiaminopimelate. DHDPS is regulated through feedback inhibition when lysine binds at a location distinct from the reactive site. The mechanism by which lysine remotely disrupts catalysis is not well understood. A clear understanding of how the natural inhibitor, lysine, binds to DHDPS and what effects this has on the machinery of the enzyme will be invaluable for development of novel antibiotic leads. Analysis of DHDPS crystals with and without inhibitors has revealed structural changes that appear to link the allosteric and active sites. Our ongoing research examines the validity of observed structural changes in the mechanism of allosteric inhibition.