-
T V Fedorova,
A M Chulkin,
E A Vavilova,
I G Maisuradze,
A A Trofimov,
I N Zorov,
V P Khotchenkov,
K M Polyakov,
S V Benevolensky,
O V Koroleva, V S Lamzin
[show abstract]
[hide abstract]
ABSTRACT: The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.
Biochemistry (Moscow) 10/2012; 77(10):1190-8. · 1.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The three-dimensional structure of the complex of the enzyme SAICAR synthase with the product of the enzymatic reaction, SAICAR,
was solved and refined by methods of protein crystallography. The SAICAR-binding site in the active site of the enzyme was
found. The amino-acid residues providing the binding of the reaction product with the protein were revealed. These residues
were compared with those involved in the substrate binding in the complex with AICAR and succinic acid studied earlier.
Crystallography Reports 09/2006; 51(5):824-827. · 0.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2006; 62(Pt 3):215-7. · 0.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Methods for automated identification and building of protein-bound ligands in electron-density maps are described. An error model of the geometrical features of the molecular structure of a ligand based on a lattice distribution of positional parameters is obtained via simulation and is used for the construction of an approximate likelihood scoring function. This scoring function combined with a graph-based search technique provides a flexible model-building scheme and its application shows promising initial results. Several ligands with sizes ranging from 9 to 44 non-H atoms have been identified in various X-ray structures and built in an automatic way using a minimal amount of prior stereochemical knowledge.
Acta Crystallographica Section D Biological Crystallography 01/2005; 60(Pt 12 Pt 1):2230-9. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The relation between a Gaussian perturbation of the atomic positional parameters and the average squared structure-factor amplitude is presented. Using an error-dependent radial distance distribution of an atomic protein model, it can be shown that the Debye effects diminish exponentially as a function of increasing positional errors. These relations can be used to estimate the quality of an atomic model and the corresponding phases. The limiting case of equal atoms with an infinitely large coordinate error results in the classical Wilson model.
Acta Crystallographica Section D Biological Crystallography 03/2004; 60(Pt 2):220-6. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The analytical expression for the distribution of an interatomic distance resulting from a known error-free distance and a Gaussian perturbation of the atomic coordinates is presented. This is used to estimate the coordinate error on the basis of known geometric features of protein models via the nearest-neighbour or the radial distance distribution. A simple relation is presented that describes the dependence of the map correlation on the positional error of the protein model, the resolution of the X-ray data and the overall atomic displacement parameter. The distribution of geometrical features and the relation between the map correlation and the positional error can be used in assisting the decision-making process during automated model-building procedures.
Acta Crystallographica Section D Biological Crystallography 01/2004; 59(Pt 12):2104-13. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Crystals of Saccharomyces cerevisiae inorganic pyrophosphatase suitable for X-ray diffraction study were grown by cocrystallization of the enzyme with cobalt
chloride and imidodiphosphate. Saccharomyces cerevisiae is a metal-dependent enzyme which catalyzes hydrolysis of inorganic pyrophosphate to orthophosphate. The three-dimensional
structure of this enzyme was solved by the molecular-replacement method and refined at 1.8 Å resolution to an R factor of 19.5%. Cobalt and phosphate ions were revealed in the active centers of both identical subunits (A and B) of the pyrophosphatase molecule. In subunit B, a water molecule was found between two cobalt ions. It is believed that this
water molecule acts as an attacking nucleophile in the enzymatic cleavage of the pyrophosphate bond. It was demonstrated that
cobalt ions and a phosphate group occupy only part of the potential binding sites (two chemically identical and crystallographically
independent subunits have different binding sites). The arrangement of ligands and the structure of the nucleophile-binding
site are discussed in relation to the mechanism of action of the enzyme and the nature of the metal activator.
Crystallography Reports 01/2003; 48(6):953-958. · 0.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively. In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)). This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging. The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent. To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed. The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described.
Journal of Molecular Biology 12/2001; 314(3):633-45. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The aim of ARP/wARP is improved automation of model building and refinement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to refine and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and refining the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible refinement of otherwise difficult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A, but for map improvement considerable advances over more conventional refinement techniques are evident even at 3.2 A spacing.
Acta Crystallographica Section D Biological Crystallography 11/2001; 57(Pt 10):1445-50. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex.
The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase.
A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.
Structure 09/2001; 9(8):725-38. · 6.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The three-dimensional structures of two enzyme-substrate complexes of SAICAR synthase from the yeast Saccharomyces cerevisiae with adenosinetriphosphate (ATP) prepared under different conditions were studied by X-ray diffraction analysis and then
refined. An enzyme molecule was shown to contain two binding sites of ATP. One of these sites is located in the central cavity
of the enzyme molecule and apparently binds the ATP molecule directly involved in the enzymatic reaction. In the complexes,
the phosphate groups of ATP occupying this site adopt different conformations depending on the Mg2+ concentration. The functional role of the second binding site located at a distance of approximately 15 Å from the first
site away from the central enzyme cavity has not been understood as yet. It might be that the second site perform the regulatory
role in enzyme functioning.
Crystallography Reports 06/2001; 46(4):620-625. · 0.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Atomic (1 A) resolution x-ray structures of horse liver alcohol dehydrogenase in complex with NADH revealed the formation of an adduct in the active site between a metal-bound water and NADH. Furthermore, a pronounced distortion of the pyridine ring of NADH was observed. A series of quantum chemical calculations on the water-nicotinamide adduct showed that the puckering of the pyridine ring in the crystal structures can only be reproduced when the water is considered a hydroxide ion. These observations provide fundamental insight into the enzymatic activation of NADH for hydride transfer.
Journal of Biological Chemistry 04/2001; 276(12):9316-21. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A goal of structural biology--and of structural genomics in particular--is to improve the underlying methodology for high-throughput determination of three-dimensional structures of biological macromolecules. Here we address issues related to the development, automation and streamlining of the process of macromolecular X-ray crystal structure solution.
Natural Structural Biology 12/2000; 7 Suppl:978-81.
-
Acta Crystallographica Section D Biological Crystallography 12/2000; 56(Pt 11):1510-1. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A significant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the flash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modified concentrations of the components. The crystal was then flash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A, from 1.8 to 1.15 A.
Acta Crystallographica Section D Biological Crystallography 06/2000; 56(Pt 5):595-603. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The crystal structures of two forms of the enzyme dimanganese catalase from Thermus Thermophilus (native and inhibited by chloride) were studied by X-ray diffraction analysis at 1.05 and 0.98 Å resolution, respectively.
The atomic models of the molecules were refined to the R factors 9.8 and 10%, respectively. The three-dimensional molecular structures are characterized in detail. The analysis of
electron-density distributions in the active centers of the native and inhibited enzyme forms revealed that the most flexible
side chains of the amino acid residues Lys162 and Glu36 exist in two interrelated conformations. This allowed us to obtain
the structural data necessary for understanding the mechanism of enzymatic activity of the dimanganese catalase.
Crystallography Reports 12/1999; 45(1):105-116. · 0.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We demonstrate with two examples the success and potential of recent developments in x-ray protein crystallography at ultra high resolution. Our preliminary structural analyses using diffraction data collected for the two proteins crambin and savinase show meaningful deviations from the conventional independent spherical atom approximation. A noise-reduction averaging technique enables bonding details of electron distributions in proteins to be revealed experimentally for the first time. We move one step closer to imaging directly the fine details of the electronic structure on which the biological function of a protein is based.
Journal of Biological Chemistry 08/1999; 274(30):20753-5. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.
Natural Structural Biology 06/1999; 6(5):458-63.
-
[show abstract]
[hide abstract]
ABSTRACT: The pharmacological properties of a cyclomaltoheptaose (beta-cyclodextrin) series of adamantane-group-bearing compounds that exhibit potent antibacterial activity have been studied, both isolated and in complex with beta-cyclodextrins (betaCDs). In this work, the structure of the bromide salt of 2-(3-dimethylaminopropyl)-tricyclo[3.3.1.1(3,7)]decan-2-ol(A DM-10) complexed with betaCD and ten water molecules was studied in the solid state by X-ray crystallography and in solution by NMR spectroscopy. X-ray crystallographic studies of the complex were performed both at room and cryogenic temperatures. The long aliphatic chain of ADM-10 adopts a single conformation at low temperature in contrast to what is observed at room temperature, where two side chain conformations are seen. Both NMR and X-ray crystallography studies indicate that the adamantane moiety of ADM-10 is buried in the betaCD cavity. Chemical shifts in NMR experiments can be explained on the basis of the crystal structure of the complex.
Carbohydrate Research 05/1999; 317(1-4):19-28. · 2.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: X-ray diffraction data have been collected at both low (120 K) and room temperature from triclinic crystals of hen egg-white lysozyme to 0.925 and 0.950 A resolution, respectively, using synchrotron radiation. Data from one crystal were sufficient for the low-temperature study, whereas three crystals were required at room temperature. Refinement was carried out using the programs PROLSQ, ARP and SHELXL to give final conventional R factors of 8.98 and 10.48% for data with F > 4sigma(F) for the low- and room-temperature structures, respectively. The estimated r.m.s. coordinate error is 0.032 A for protein atoms, 0.050 A for all atoms in the low-temperature study, and 0.038 A for protein atoms and 0.049 A for all atoms in the room-temperature case, as estimated from inversion of the blocked least-squares matrix. The low-temperature study revealed that the side chains of 24 amino acids had multiple conformations. A total of 250 waters, six nitrate ions and three acetate ions, two of which were modelled with alternate orientations were located in the electron-density maps. Three sections of the main chain were modelled in alternate conformations. The room-temperature study produced a model with multiple conformations for eight side chains and a total of 139 water molecules, six nitrate but no acetate ions. The occupancies of the water molecules were refined in both structures and this step was shown to be meaningful when assessed by use of the free R factor. A detailed description and comparison of the structures is made with reference to the previously reported structure refined at 2.0 A resolution.
Acta Crystallographica Section D Biological Crystallography 07/1998; 54(Pt 4):522-46. · 12.62 Impact Factor
