bluuebunny/crossref_metadata_2025 huggingface dataset:
Parametric Rietveld refinement applied to dehydration of sodium naproxen
Authors: Sovago Ioana , Bond Andrew , van de Streek Jacco , Thomas Diana
Published: November 2015

The identification of the hydration and dehydration transformations of drugs is vital to establish stable pharmaceutical components. Our aim is to monitor the kinetics of hydration and dehydration processes in pharmaceutical solids, and to relate these to the molecular-level crystal structures. One of the primary tools to achieve this is parametric Rietveld refinement. The dehydration of two dihydrate polymorphs of the non-steroidal anti-inflammatory drug (NSAID) sodium naproxen was monitored using synchrotron powder X-ray diffraction measurements at Beamline I711, MAX IV Laboratory, Lund University. Diffraction patterns were measured in the range 300-400 K at 1 K increments. Both polymorphs dehydrate to form identical monohydrate then anhydrous phases. Independent Rietveld refinements were initially performed for each unique phase in order to establish initial values for the parametric refinement. The refinements were performed using TOPAS-Academic [1]. The structures are molecular and both dihydrate polymorphs display pseudosymmetry, thereby requiring an extensive set of restraints. One of the key advantages of the parametric Rietveld refinement is the possibility to introduce algebraic equations that describe the evolution of various parameters [2]. The kinetics of the dehydration processes were monitored using a sigmoid function applied to the scale factors of the various phases (see Figure). The evolution of the unit-cell parameters and atomic displacement parameters were also treated parametrically, and the influence of using multiple coordinate sets (one model per temperature) or a single common coordinate set for each phase was examined. One dihydrate polymorph shows a smooth and continuous transition to the monohydrate, whereas the other polymorph shows an abrupt transition. These differences are linked to the existence of topotactic or non-topotactic chemical transformations between the dihydrate polymorphs and the monohydrate phase.
Processing Extremely Large Powder Diffraction Datasets Using The Rietveld Method
Authors: Wragg David , O'Brien Matthew , Di Michiel Marco , Lønstad Bleken Francesca , Fjellvåg Helmer , Olsbye Unni
Published: November 2015

Modern in situ synchrotron powder diffraction experiments can produce massive volumes of data which are of suitable quality for real structural information to be extracted. Parametric Rietveld refinement (Stinton, 2007) is an ideal method for dealing with such datasets as a huge number of diffraction patterns can be processed in parallel while the number of refined parameters is reduced by linking between scans. The time saving compared to earlier sequential methods of refinement using batch files is very significant with processing times being reduced from weeks to a few hours. The stability of the parametric method allows not only extraction of information from data with very weak trends but also refinement of entire slices of a tomographic map including the regions with zero diffraction. The power of the technique will be illustrated by examples from reactor scanning experiments with high time resolution (Wragg, 2012, 2013) as well as more conventional in situ powder diffraction and operando experiments combining diffraction with mass spectrometry. The extraction of structural information from complete tomographic datasets and reconstructions with real structural parameters will also be demonstrated. The figure shows time and space resolved c-axis data for the SAPO-34 catalyst during methanol to olefin conversion, together with mass spectrometry data collected during the experiment.
Mode parameterization of structures with very low symmetry: PZT and magnetite
Authors: Perez-Mato J. Manuel , Kocsis Balazs , Tasci Emre , Aroyo Mois
Published: November 2015

The parameterization of distorted structures in terms of symmetry modes is an effective and efficient method for both their description and refinement [1]. A basis of symmetry-adapted modes transforming according to irreducible representations not only provides a hierarchical division of the degrees of freedom consistent with the mechanism at the origin of the distorted phase, but it allows the avoidance of false refinement minima, typical of highly pseudo-symmetric phases. A reduction of the number of free parameters by setting to zero negligible marginal modes is also possible. The mode description is nowadays easily applicable through freely available programs [2,3], while direct single crystal and powder diffraction refinements under this parameterization are possible combining these programs with some of the most popular refinement codes. The mode description is especially effective when dealing with distorted structures of very low symmetry compared with that of the parent phase. In these cases, the hierarchy between strong primary modes and weak marginal ones is specially pronounced, minimizing the role of many secondary modes. The physical origin of each primary distortion is usually a set of unstable degenerate normal modes. This introduces correlations among the different phases in the phase diagram that become patent in a mode description and can be used both to characterize the evolution of the relevant order parameters and as a stringent test of proposed structural models. Furthermore, the fact that each of the primary mode distortions is basically associated with the activity (instability) of a single normal mode can yield a "single mode" signature in the mode decomposition, which represents a set of subtle additional structural constrains beyond conventional crystallography. We will illustrate these considerations using the examples of the monoclinic phases of ferroelectric PbZr1-xTixO3 (PZT) and the Verwey phase of magnetite.
Using the Knowledge from Every Organic Crystal Structure Ever Published
Authors: Groom Colin , Ward Suzanna , Feeder Neil , Pidcock Elna , Wood Peter , Galek Peter
Published: November 2015

The crystallographic community has done something remarkable and almost unique in science. It has operated in such a way that the data generated in virtually every experiment reported in a publication is available to all. This data, in the form of individual crystal structures, is valuable not just in itself, but as a collection. To fully exploit the results of a new structure determination, we never analyse a single structure, we analyse it in the context of every previous crystal structure. Our knowledge of molecular geometry and molecular interactions derived from these structural databases is put to routine use in almost every field of chemistry. This presentation will specifically highlight what we can learn from the world's database of small molecule crystal structures and demonstrate how we can apply that knowledge not just to increase our understanding in structural chemistry, but in structural biology too.
Transforming biomedical and structural data into information and knowledge
Authors: Zimmerman Matthew , Cooper David , Cymborowski Marcin , Konina Krzysztof , Grabowski Marek , MacLean Elizabeth , Minor Wladek
Published: November 2015

The LabDB laboratory information management system (LIMS) tracks, organizes and analyzes data from chemical and solution management, protein production, crystallization, diffraction, structure solution, and in vitro biochemical and biophysical experiments. The system is comprised of multiple modules specialized for different tasks, such as the Xtaldb system for crystallization or the hkldb module of the HKL-3000 suite for diffraction data collection and structure solution. The biochemical/biophysical experiments tracked by LabDB include spectrophotometric binding and kinetics, thermal shift binding, isothermal titration calorimetry (ITC) and protein quantitation. These tools associate functional and structural experiments, for example, for selecting likely substrates for co-crystallization and soaking experiments. Whenever possible, the system harvests data with no or minimal user intervention from laboratory hardware. Devices that may connect to or import data into LabDB include crystal observation (Rigaku Minstrel HT and Formulatrix Rock Imager), liquid handling (Formulatrix Rock Maker and Emerald Opti-Matrix Maker), chromatography (GE Healthcare AKTA), quantitation (Caliper LabChip GX II and Bio-Rad Gel Doc EZ), RT-PCR (Applied Biosystems 7900HT and Bio-Rad C1000/CFX96) and ITC (MicroCal iTC-200) systems. LabDB is used by two high-throughput PSI:Biology centers (MCSG and NYSGRC) as well as other major NIH consortia (the Center for Structural Genomics of Infectious Diseases and the Enzyme Function Initiative), and track millions of experiments on tens of thousands of targets.[1] The system also provides extensive data mining and analysis tools for translating raw experimental data into information and knowledge. We present examples of analyses generated by the system useful in designing new experiments.
Virus Maturation and the VIPERdb Virus Structural Database
Authors: Johnson John
Published: November 2015

VIPERdb (Carrillo-Tripp, M., Shepherd, C. M., Borelli, I. A., Venkataraman, S., Lander, G., Natarajan, P., Johnson, J. E., Brooks, C. L., 3rd, and Reddy, V. S. 2009. VIPERdb2: an enhanced and web API enabled relational database for structural virology. Nucleic Acids Res 37:D436-42) is a database for icosahedral virus capsid structures. The emphasis of the resource is on providing data from structural and computational analyses on these systems, as well as high quality renderings for visual exploration. The web site includes powerful search utilities and useful database interface tools. Here we use VIPERdb to address the dynamic character of a virus by relating cryoEM based models of maturation intermediates and associated variance analysis to the high resolution coordinates in VIPERdb for the T=4 Nudaurelia Capensis ω Virus. The outcome is a structure-based description of the maturation energy landscape at near atomic resolution (Tang, J., Kearney, B., Wang, Q., Doerschuk, P. C., Baker, T. S., and Johnson, J. E. 2013. Geometric and Dynamic Analyses of Nudaurelia capensis ω Virus Maturation Reveal the Energy Landscape of Particle Transitions. J. Mol. Recog. Accepted for publication).
The Structural Basis for BVMO Substrate Profile and Stereospecificity
Authors: Yachnin Brahm , McEvoy Michelle , MacCuish Roderick , Morley Krista , Mittermaier Anthony , Lau Peter , Berghuis Albert
Published: November 2015

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are able to catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymes have attracted considerable attention as potential industrial biocatalysts. The interest in these enzymes has led to a desire to be able to rationally design them for tailored biocatalytic applications. While recent years have seen the publication of a number of crystal structures (1-3), we have been lacking a structure of a BVMO that has its native substrate or product bound in a conformation that will allow the determination of substrate specificity and stereospecificity. Without such a structure, progress towards tailored BVMOs has been hampered. We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO) with its lactone product, ε-caprolactone, bound. These structures place the lactone in an ideal position for the determination of its substrate specificity and stereospecificity. These structures have provided us with a better understanding of the structural basis for substrate binding, paving the way for the rational design of tailored BVMOs. At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies to better understand the dynamic nature of the enzyme. These studies have allowed us to explain the relationship between the various crystallized states of BVMOs and their complex, fourteen step enzyme mechanism.
Structural basis for the biosynthesis of the CN ligand of [NiFe] hydrogenase
Authors: Watanabe Satoshi , Tominaga Taiga , Matsumi Rie , Atomi Haruyuki , Imanaka Tadayuki , Miki Kunio
Published: November 2015

[NiFe] hydrogenases carry a NiFe(CN)2CO center at the active site, catalyzing the reversible H2oxidation. The complex NiFe center is biosynthesized and inserted into the enzyme by six specific maturation proteins: Hyp proteins (HypABCDEF). HypE and HypF are involved in biosynthesis of cyanide ligands, which are attached to the Fe atom in the NiFe center. First, HypF catalyzes a transfer reaction of the carbamoyl moiety of carbamoylphosphate to the C-terminal cysteine residue of HypE. Then, HypE catalyzes an ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. In order to elucidate the catalytic mechanism of HypE, we have determined the crystal structures of the carbamoylated and cyanated states of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53 Å and 1.64 Å resolution, respectively [1]. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. A conserved glutamate residue (Glu272) is close to the thiocarboxamide nitrogen atom of Cys338. However, the configuration of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. Interestingly, a conserved arginine residue makes close contact with Lys134 and lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.
A preliminary study of Dihydroorotase from Methanococcus jannaschii
Authors: Singh Aditya , Colaneri Michael , Vitali Jacqueline
Published: November 2015

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to form L-dihydroorotate in the third step of de novo pyrimidine biosynthesis. It is a Zinc metalloenzyme and a member of the aminohydrolase superfamily. There are two classes of the enzyme. Class I, typically ~45 kDa, is found in higher organisms, bacteria and yeast. Class II, typically ~38 kDa, is found in bacteria and fungi. Some organisms have multiple DHOase sequences. The M. jannaschii pyrC gene coding for DHOase was subcloned and expressed in E. coli. Protein purification consisted of ammonium sulfate precipitation, heat treatment at 850C, and phenyl-sepharose hydrophobic interaction chromatography. The protein was confirmed in the SDS gel using Liquid Chromatography-Mass Spectrometry (Proteomics Laboratory, Lerner Research Institute, Cleveland, OH). Size Exclusion Chromatography-Laser Light Scattering (Keck Biotechnology Laboratory, Yale University, New Haven, CT) indicated that the protein is a monomer in solution with a molecular weight of 47 kDa. A model constructed with the I-TASSER server (Zhang, 2008) suggested that the binding site contains only one Zn ion per monomer coordinated by the conserved His56, His58 and Asp302. Asp146 is further away and does not coordinate with the Zn ion. According to the mass spectrometry analysis, the protein does not contain a carboxylated lysine. Our progress on this study will be presented. Acknowledgements: We thank Dr. Belinda Willard (Lerner Research Institute) for the LC-MS and Dr. Ewa Folta-Stogniew (Yale University) for the SEC- LS analysis. The presentation was supported in part by a graduate faculty travel award and by a contribution from the Physics Department at Cleveland State University.
Complex structure of NADPH-cytochrome P450 reductase and heme oxygenase-1
Authors: Sugishima Masakazu , Sato Hideaki , Higashimoto Yuichiro , Harada Jiro , Wada Kei , Fukuyama Keiichi , Noguchi Masato
Published: November 2015

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to FAD in CPR, then to FMN in CPR, and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, ``closed-open transition'' of CPR is indispensable because FMN in the closed conformation of CPR is covered by FAD-binding domain, thus FMN is not exposed to the surface in the closed conformation. Recently, Hamdane et al. determined the crystal structures of a hinge-shortened rat CPR variant (ΔTGEE), which favors an open conformation [1]. In the open conformation of CPR, FMN is exposed to the surface, thus this conformation appears to be favorable to interact with the redox partners, though no complex structure of CPR and its redox partner has been determined. Here, we demonstrate that ΔTGEE makes a stable complex with heme-rat HO-1 (rHO-1) complex and can support HO reaction, though its efficiency is extremely limited. Further we determine the crystal structure of ΔTGEE in complex with heme-rHO-1 at 4.3 Å resolution [2]. X-ray scattering and biochemical data suggest that the complex structure of ΔTGEE and heme-rHO-1 is similar to that of wild type CPR and heme-rHO-1. Distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme. On the other hand, FAD is far from FMN and heme, indicating that the ``closed-open transition'' of CPR is required for electron transfer from FAD to FMN.
Characterization of transglutaminase homologues found in andrimid biosynthesis
Authors: Reimer Janice , Schmeing T
Published: November 2015

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize products with diverse structures and activities ranging from antibiotics to industrial solvents. They are arranged into an assembly line of modules where each module is responsible for incorporating a specific monomer into the final nonribosomal peptide (NRP). Diversity in NRPs stems from the fact that NRPSs utilize not only the 20 proteinogenic amino acids, but also include nonproteinogenic amino acids, fatty acids, and alpha-hydroxy acids as building block substrates. Andrimid is a NRP antibiotic that inhibits membrane biosynthesis by blocking bacterial acetyl coenzyme A carboxylases. It is synthesized in a hybrid NRPS-polyketide synthase (NRPS-PKS) using a fatty acid, phenylalanine, valine, and glycine. A remarkable feature of this synthetic system is that instead of a normal condensation domain, it uses two atypical free-standing proteins with homology to transglutaminases to catalyze the formation of the first and second amide bonds. We are characterizing the action of transglutaminase homologues (TGH) in andrimid synthesis using biochemical assays and X-ray crystallography. Initial investigations of the andrimid biosynthetic cluster found in Panteao agglomerans focused on the TGH, AdmF, which catalyzes the formation of the first amide bond. Crystallization trials have been initiated on AdmF in its apo form and in complex with its interacting binding partner, the peptide carrier protein domain AdmI. To date, only a few andrimid producing bacteria have been discovered. Using genome mining, a biosynthetic cluster homologous to the andrimid biosynthetic cluster found in Panteao agglomerans was identified in Vibrio coralliilyticus. The two TGHs, CoraF and CoraS, were cloned, expressed and purified, and crystallization trials are underway. Our progress in biochemical and biophysical characterization of AdmF, CoraF, and CoraS will be presented.
Novel intersubunit active site of arcchaeal N,N′-diacetylchitobiose deacetylase
Authors: Nakamura Tsutomu , Niiyama Mayumi , Hashimoto Wakana , Ikegami Takahisa , Koma Daisuke , Ohmoto Takashi , Morita Junji , Uegaki Koichi
Published: November 2015

N,N′-diacetylchitobiose deacetylase (Dac) is involved in the archaea-specific chitinolytic pathway. In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on Dacs from P. horikoshii (Ph-Dac) and P. furiosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were thermostable up to 950C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer comprised of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic zinc situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. These factors give the archaeal Dacs unprecedented active site architecture as a zinc-dependent deacetylase.
Structure and specificity of novel aminopeptidase from marine sediment Archaea
Authors: Michalska Karolina , Steen Andrew , Chhor Gekleng , Fayman Katlyn , Endres Michael , Babnigg Gyorgy , Lloyd Karen , Jedrzejczak Robert , Joachimiak Andrzej
Published: November 2015

The Earth's microbial diversity remained largely unexplored until recent developments in DNA sequencing. Novel methods enabled us to access genomic information of uncultured microbial organisms and create hypotheses about their metabolic capabilities. These predictions primarily rely on the sequence similarity between a novel protein and characterized proteins. Such an approach introduces a "culture" bias: the well-understood proteins come from a set of laboratory-grown bacteria, while novel microbial proteins are obtained from a variety of environments, including the most extreme. One such niche is ocean sediment – an unexplored ecosystem that plays important roles in geochemical cycles. Single-cell genomics targeting sedimentary populations identified four new archaeons encoding putative intra- and extra-cellular proteases [1]. This discovery suggests that heterotrophic marine Archaea evolved to degrade detrital proteins and might contribute to global carbon cycling. The novel proteases share some sequence similarity with well-known protein-degrading enzymes, but generally are distant homologs. Thus, functional screening is necessary to validate sequence-based predictions. One of the proteases shares sequence similarity with S15 peptidases, cocaine esterases and α-amino acid ester hydrolases (AEH). Phylogeny indicates that the gene is of bacterial origin. Enzymatic assays reveal α-aminopeptidase activity towards dipeptides with a preference for a small, L-configured hydrophobic residue at the N-terminus. The crystal structure shows a homotetrameric, self-compartmentalizing enzyme with four independent active sites localized inside the oligomeric assembly accessible from the internal channel. The active site contains a serine protease triad (Ser-His-Asp) and a cluster of negatively charged residues that bind the N-terminal NH3+ group of the substrate molecule. Therefore, the observed activity suggests that the enzyme (designated as AP TA1) may act on di- or tri-peptides produced during extracellular degradation and subsequently imported to the cell. As a close homolog of AEHs, it is also possible that AP TA1 might participate in the synthesis of yet-to-be-discovered secondary metabolites. Supported by NIH GM094585, DOE/BER DE-AC02-06CH11357 & C-DEBI 36202823 & 157595.
Structural characterisation of a polyphosphate kinase
Authors: Loasby Alice , Roach Peter , Oyston Petra
Published: November 2015

When deprived of nutrients bacteria undergo what is referred to as the `stringent response'. During the stringent response, the cell induces the expression of genes to cope with stress and starvation and diverts resources away from cell growth and division. This involves altering the cellular levels of the signalling molecules: ppGpp, pppGpp and inorganic polyphosphate (polyP)[1]. This is controlled by four enzymes; Polyphosphate kinase (Ppk), Exopolyphosphatase (Ppx), RelA and SpoT. Therefore, modulation of these enzymes is an attractive method for targeting pathogenic bacteria such as Francisella tularensis (F. tularensis), the causative agent of tularemia. FtPpk transfers phosphate from polyP to ADP to generate ATP, a reaction that is fully reversible. The importance of FtPpk in infection has been demonstrated in knockout mutants which resulted in defective growth of F. tularensis in macrophages[2]. Mutagenesis in other pathogenic bacteria has yielded attenuated mutants, suggesting an important role for Ppk in a broad spectrum of bacterial species[3]. To maximise our understanding of FtPpk, our aim was to obtain co-crystals of the enzyme and substrates. Isothermal Titration Calorimetry (ITC) was used to measure the binding of polyP and ADP to FtPpk as independent substrates. FtPpk binds ADP very weakly or not at all in the absence of polyP. FtPpk binds polyP in an exothermic reaction with a relatively high affinity (0.385 µM) in the absence of ADP. Co-crystals of FtPpk with polyP and ADP have been obtained and optimised to diffract to 2.0 Å, identifying a potential binding site for polyP. A non-hydrolysable analogue of ATP has been chemically synthesised to allow co-crystallisation experiments.
Crystal structure of the M14R mutant of oryctin in complex with trypsin
Authors: Liu Desheng , Suzuki Tatsuya , Horita Shoichiro , Kawai Takeshi , Ishibashi Jun , Yamakawa Minoru , Nagata Koji , Tanokura Masaru
Published: November 2015

Oryctin is a 66-amino-acid protein purified from the larval haemolymph of the coconut rhinoceros beetle Oryctes rhinoceros, which shows no sequence similarity to any other protein known. We determined the solution NMR structure of oryctin, and found that oryctin had a similar backbone fold to the turkey ovomucoid domain 3, OMTKY3, a Kazal-type serine protease inhibitor [1]. Based on the structural similarity, we tested the serine protease inhibitory activity of oryctin, and found that oryctin does inhibit some serine proteases, such as α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase [1]. However, oryctin cannot inhibit trypsin at all. In this study, we have introduced point mutations to the putative inhibition loop of oryctin to obtain oryctin mutants that can inhibit trypsin. Then, we have solved the crystal structure of such an oryctin mutant, M14R-oryctin with a Ki value of 3.410.8 nM, in complex with trypsin to reveal how it binds to and inhibits trypsin. As predicted, the putative inhibition loop lay on the substrate binding cleft of trypsin. Particularly, the side chain of R14 fit into the S1 pocket of trypsin by forming hydrogen/ionic bonds with D191, S192 and G216 at the bottom of the S1 pocket and G195, D196, S197 and S212 at its entrance. In addition, R65 located in the C-terminal α-helix of M14R-oryctin formed hydrogen bonds with S40 and F44 of trypsin. The latter interaction, which is unique to oryctin, enhances its binding affinity to trypsin.
Optimizing enzyme behaviour through protein engineering
Authors: Lazidou Katerina , Tzortzis George , Charalampopoulos Dimitrios , Watson Kimberly
Published: November 2015

Galactooligosaccharides (GOS) constitute an important class of prebiotic compounds used by the food industry as active ingredients with potential health benefits. GOS are enzymatically produced from lactose using β-galactosidases through a reaction known as transgalactosylation. Many studies have been conducted in an attempt to increase GOS yields by controlling the reaction conditions using β-galactosidases from a range of microorganisms. In this study, we have used high-throughput protein engineering for two GOS producing β-galactosidases, BbgIII and BbgIV from Bifidobacterium bifidum in an effort to enhance transgalactosylation activity (over hydrolysis) thus favouring GOS synthesis. A total of 36 and 11 C- and N-terminus deletion mutants were designed for BbgIII and BbgIV, respectively. The mutant constructs ranged from highly active to completely inactive enzymes. Selected constructs were tested for their transgalactosylation activity. An increase ranging between 5 and 10% (of total carbohydrates) was obtained with the mutant enzymes. Additionally, up to 2-fold increase in the higher degree of polymerization of GOS products was observed for selected mutants compared to the native enzyme. Structure determination of two highly active constructs at 2.0 Å resolution indicated that truncations affected the oligomeric state of the enzymes, which may have implications for activity.
Crystal structure of archaeal phosphopantothenate synthetase
Authors: Kita Akiko , Kishimoto Asako , Ishibashi Takuya , Tomita Hiroya , Yokooji Yuusuke , Imanaka Tadayuki , Atomi Haruyuki , Miki Kunio
Published: November 2015

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes, pantothenate synthetase and pantothenate kinase, to convert pantoate to 4'-phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase (PoK) and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea[1]. In archaea, pantoate is phosphorylated by PoK to produce 4-phosphopantoate (PPo), and then condensation of PPo and β-alanine is catalyzed by PPS, generating 4'-phosphopantothenate. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with ATP, and ATP and 4-phosphopantoate (PPo). PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact 2-fold symmetry, displaying 40-130distortion. Two active sites in PPS dimer are located near the rotation axis. Due to the asymmetricity of PPS dimer molecule, two active sites in PPS dimer are not equivalent. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on the structures of PPS, PPS/ATP complex, and PPS/ATP/PPo complex, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate (PPA), which is thought to be a reaction intermediate.
Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase
Authors: Gerlits Oksana , Das Amit , Tian Jianhui , Keshwani Malik , Taylor Susan , Waltman Mary Jo , Langan Paul , Heller William , Kovalevsky Andrey
Published: November 2015

Protein kinases are involved in a number of cell signaling pathways. They catalyze phosphorylation of proteins and regulate the majority of cellular processes (such as growth, differentiation, lipid metabolism, regulation of sugar, nucleic acid synthesis, etc.). Chemically, protein kinases covalently transfer the gamma-phosphate group of a nucleoside triphosphate (e.g. ATP) to a hydroxyl group of a Ser, Thr or Tyr residue of substrate protein or peptide. The reaction involves moving hydrogen atoms between the enzyme, substrate and nucleoside. The unanswered question is whether the proton transfer from the Ser residue happens before the phosphoryl transfer using the general acid-base catalyst, Asp166, or after the reaction went through the transition state by directly protonating the phosphate group. To address this key question about the phosphoryl transfer, we determined a number of X-ray structures of ternary complexes of catalytic subunit of cAMP-dependent protein kinase (PKAc) with various substrates, nucleotides and cofactors. Importantly, we were able to trap and mimic the initial (Michaelis complex) and final (product complex) stages of the reaction. The results demonstrate that Mg2+, Ca2+, Sr2+, and Ba2+ metal ions bind to the active site and facilitate the reaction to produce ADP and a phosphorylated peptide. The study also revealed that metal-free PKAc can facilitate the phosphoryl transfer reaction; a result that was confirmed with single turnover enzyme kinetics measurements. Comparison of the product and the pseudo-Michaelis complex structures, in conjunction with molecular dynamics simulations, reveals conformational, coordination, and hydrogen bonding changes that help further our understanding of the mechanism, roles of metals, and active site residues involved in PKAc activity.
Substrate distortion in the catalysis of orotidine monophosphate decarboxylase
Authors: Fujihashi Masahiro , Ishida Toyokazu , Kuroda Shingo , Mito Kazuya , Kotra Lakshmi , Pai Emil , Miki Kunio
Published: November 2015

One way for enzymes to affect reactions they catalyze is through transition state stabilization. Another factor to be considered is the contribution of substrate distortion, although it has been thoroughly described for only a few enzymes. We have a longstanding interest in the reaction mechanism of orotidine monophosphate decarboxylase (ODCase) and determined various crystal structures bound with distorted substrates at around 1.5 Å resolution. The enzyme is known as one of the most proficient enzymes, which accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to form uridine 5'-monophosphate (UMP) by 17 orders of magnitude. One argument against the contribution of substrate distortion to the ODCase reaction is the weak affinity of UMP. The distortions observed so far all appear at the C6-substituent of the pyrimidine ring, which corresponds to the carboxylate of OMP. Since the carboxylate is removed by the reaction, the product UMP should bind more tightly to ODCase than OMP, if the distortion of C6-substituent contributes to the catalysis. In order to investigate this inconsistency, we determined the crystal structure of ODCase with UMP at atomic resolution (1.03 Å). The structure showed an unfavorable interaction between UMP and the catalytic residue K72, an interaction considered to be absent in the OMP complex. Surface plasmon resonance analysis indicated that UMP binds stronger to the K72A mutant than to the wild-type enzyme by 5 orders of magnitude. These analyses invalidate the argument against a contribution of substrate distortion to ODCase catalysis. Finally, we estimated how much the distortion contributes to the catalysis using computational simulation methods. The results indicated that 10-15% decrease of the ΔΔG‡ value is contributed by substrate distortion.
Structural and mechanistic studies of human chitinase
Authors: Fadel Firas , Zhao Yuguang , Cousido-Siah Alexandra , Howard Eduardo , Mitschler André , Podjarny Alberto
Published: November 2015

Chitinases are enzymes that hydrolyze chitin, a glucosamine polymer synthesized by lower organisms for structural purposes [1]. While humans do not synthetize chitin, they express two active chitinases, Chitotriosidase (hCHIT1) and Acidic Mammalian Chitinase (hAMCase). Both enzymes attracted attention due to their upregulation in immune system disorders [2,3]. They consist of a catalytic domain of 39 kDa and a chitin binding domain, joined by a hinge. The active site shows a cluster of three conserved acidic residues, E140, D138 and D136, linked by H-bonds, where D138 and E140 are involved in the hydrolysis reaction [1,3]. To increase our knowledge on the catalytic mechanism of human chitinases, we conducted a detailed structural analysis on hCHIT1. For this, we have improved the X-ray resolution of the apo hCHIT1 catalytic domain to 1Å. We investigated the protonation state on the catalytic site and detected a double conformation of D138, one in contact with D136 and a second one in contact with E140. Our analysis revealed for the first time different protonation states for each conformation of D138 (fig1). Interestingly, our X-ray data suggest that the catalytic E140, supposed to donate a proton in the catalytic reaction, is deprotonated in the apo form. To gain insight on the proton transition pathway during the hydrolysis, we have solved the X-ray structure of hCHIT1 complexed with the substrate at 1.05 Å. In comparison with the apo form, this structure shows a rearrangement of the protonation states of the catalytic triad triggered by the binding of the substrate. Our results led us to suggest a new hydrolysis model involving changes in the hydrogen bond network of the catalytic triad accompanied by a flip of D138 towards D136. This contributes to protonate E140, which then donates the proton to the substrate. To confirm the role of the active site's hydrogen network, we are currently studying CHIT1 by neutron crystallography and quantum mechanics.
Helicobacter pylori Csd4 is a peptidoglycan metallocarboxypeptidase
Authors: Chan Anson , Liu Yanjie , Blair Kris , Frirdich Emilisa , Gaynor Erin , Salama Nina , Tanner Martin , Murphy Michael
Published: November 2015

The bacterial cell wall is a polymeric structure that determines the overall shape of the cell and undergoes constant remodelling during cell growth, requiring enzymes that cleave the existing peptidoglycan structure. Csd4 is an enzyme important for cell shape as deleting it in Helicobacter pylori causes the helical-shaped cells to become rod-like. Csd4 is a zinc carboxypeptidase that can cleave the tripeptide moiety found in peptidoglycan (i.e. L-Ala-γ-D-Glu-m-DAP) to release meso-diaminopimelic acid (mDAP). Structures of Csd4 were solved by X-ray crystallography up to 1.75 Å resolution in space group P212121 with zinc and substrate/product bound and contain the same unit cell dimensions. Csd4 is a monomeric enzyme with three domains: an N-terminal M14-family carboxypeptidase domain followed by two smaller domains likely important in protein-protein or protein-peptidoglycan interactions. Key interactions are observed between the protein and substrate in the active site, supporting specific substrate recognition by Csd4. A water or hydroxide molecule, which is required for catalytic activity, is also observed bound to the zinc and is poised to interact with the substrate molecule upon activation.
Understanding RET receptor tyrosine kinase ligand recognition and chemical inhibition
Authors: Burns Emily , Goodman Kerry , Kjaer Svend , Beuron Fabienne , Purkiss Andrew , Nawrotek Agata , Knowles Phillip , George Roger , Borg Annabel , Kisakye Nambozo Sara , Mouilleron Stephane , Morris Edward , McDonald Neil
Published: November 2015

The RET receptor tyrosine kinase is crucial for embryonic and adult development, with mutations in both the extracellular and kinase domains leading to several types of cancer. In order to understand the mechanisms of RET activation in more detail, we have investigated how RET interacts with its bipartite ligand comprising of a glial cell line derived neurotrophic factor (GDNF) family ligand and a GDNF family receptor (GFRalpha). To visualise this interaction, we have reconstituted two vertebrate RET ternary complexes containing both ligand and co-receptor and have determined a pseudo-atomic model for a mammalian RET ternary complex using electron microscopy. Our structures reveal the basis for ligand recognition and will be presented. As RET is a validated anti-cancer target, we are actively investigating RET chemical inhibitors in collaboration with several chemistry laboratories. We have determined structures of a diverse set of chemical scaffolds bound to RET leading to an improved RET pharmacophore based on crystallographic, biochemical and cell-based data. As current FDA-approved drugs for RET-dependent metastatic thyroid cancer suffer from off-target dose-dependent toxicity and lack of specificity, we hope our data will usefully contribute to the design of second generation RET chemical inhibitors.
Structure of muscle α-actinin: Insights into its regulation and Z-disk assembly
Authors: Ribeiro Euripedes , Pinotsis Nikos , Ghisleni Andrea , Salmazo Anita , Konarev Peter , Kostan Julius , Gkougkoulia Eirini , Aachmann Finn , Pirker Katharina , Svergun Dmitri , Gautel Mathias , Djinovic-Carugo Kristina
Published: November 2015

α-Actinin is the major component of the Z-disk, where it cross-links actin filaments from adjacent sarcomeres. It is an antiparallel dimer of 200 kDa, containing in each subunit an N-terminal actin binding domain (ABD), a central rod domain assembled from spectrin-like repeats that mediate the antiparallel assembly, and a C-terminal calmodulin-like (CaM-like) domain with 4 EF-hand motifs. Additionally to actin filaments, α-actinin binds multiple other cytoskeletal and signalling proteins. In striated muscle, the tightly defined numbers of α-actinin crosslinks between the antiparallel actin filaments at the Z-disk are organised by specific binding sites on the giant molecular blueprint of the sarcomere, titin. These titin Z-repeats contain a short, hydrophobic, α-actinin binding motif. To achieve ordered cytoskeletal assemblies, the binding properties of α-actinin must be tightly spatiotemporally regulated, in muscle α-actinin its actin and titin binding properties are regulated by phosphoinositide. Biochemical analyses led to propose previously that the α-actinin - titin interaction is regulated by an intramolecular mechanism, where the short sequence between the ABD and the rod interacts with the CaM-like domain in a pseudoligand complex, acting effectively as an intramolecular autoinhibitor. Here, we present the first complete crystallographic structure of sarcomeric human α-actinin complemented by small angle X-ray scattering data, electron-electron paramagnetic resonance, biochemical and in vivo cell biophysics studies of structure-informed mutants, which give insight into its molecular assembly and Z-disk architecture as well as into the mechanism of α-actinin function and regulation.
New flexibility/fold/interactions in the Pyruvate Dehydrogenase complex assembly
Authors: Furey William
Published: November 2015

The pyruvate dehydrogenase multienzyme complex (PDHc), is a large, macromolecular machine that converts the product of glycolysis, pyruvate, to acetyl-coenzyme A, with the overall PDHc reaction functioning as a control point in carbohydrate metabolism. Altered levels of PDHc are associated with neurological diseases including Alzheimers and Parkinsons, and because of its role in sugar metabolism, its regulatory mechanisms are targets for controlling type 2 diabetes. Inhibition of PDHc's tissue specific, regulatory kinases increase mitochondrial levels of reactive oxygen species leading to cellular apoptosis and inhibition of tumor growth, suggesting a potential for treating cancer. Two major types of complexes are found: ~4.5 megadalton, octahedral complexes containing 60 enzymatic subunits and having a cubic core; and ~9 megadalton, icosahedral complexes containing ~120 enzymatic subunits and having a dodecahedral core. Multiple copies of at least 3 key enzymatic components, E1, E2 and E3 are always present, while the larger complexes may also contain regulatory phosphatases, kinases, and non-enzymatic, E2-like proteins. The assemblies and unusual substrate channeling mechanism employed by the complexes are fascinating, as intermediates are transferred between active sites by a long, highly flexible, "swinging arm" hand-delivery system employing lipoyl domains rather than by simple diffusion. We have crystallographically analyzed the enzymatic components of PDHc from E. coli, as well as some of their binary sub-complexes. We found that: key E1-E2 interactions stabilizing the overall assembly differ substantially (displaced by over 100 angstroms!) for complexes containing homodimeric vs heterotetrameric E1 components; that the conformation of parts of homodimeric E1's is dramatically stabilized in the presence of its E2 binding partner and reveals a new fold; and that models for octahedral PDHc complexes based on icosahedral complexes are likely to be incorrect.
Distinct DNA-binding feature of the peroxide response regulator from group A streptococcus
Authors: Wang Shu-Ying , Lin Chang Sheng-Huei , Chao Shi-Yu , Hammel Michal , Nix Jay , Jeng U-Ser , Wu Jiunn-Jong
Published: November 2015

Group A streptococcus (GAS) is a significant human pathogen, renowned for its rapidly and highly desctructive ability to infect a wide variety of tissues. Clinical manifestation of GAS infection ranges from mild pharyngitis to severe life-threating disease such as necrotizing fasciitis. Unlike other gram-positive bacteria, GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species through unknown novel mechanisms. Our previous studies have discovered that the peroxide response regulator (PerR) is crucial for GAS to cope with oxidative stress and it directly regulates the expression of an iron-binding protein Dpr [1,2]. PerR is a member of Fur (ferric uptake regulator) family which is known to be dimeric, metal-binding regulators. Currently, no structural information is available to understand how the similar structures of the Fur family regulators recognize divergent DNA sequences. To study how PerR interacts with dpr promoter DNA, we have conducted a series of mutagenesis, biochemical and structural studies by combining protein crystallography and small-angle X-ray scattering (SAXS). We have determined the PerR crystal structure to 1.6 Å resolution and identified the DNA-binding residues, which suggest PerR binds to the dpr promoter through a winged-helix motif. By performing SAXS studies, we confirmed that the PerR crystal structure reflects its conformation in solution. Furthermore, SAXS analysis allowed us to resolve the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Our results have revealed the PerR-DNA interaction model and illustrated the DNA-binding mode of PerR that is distinct from all other regulators in Fur family [3].