Elucidation of Crystal Packing by X-ray Diffraction and Freeze-etching Electron Microscopy. Studies on GTP Cyclohydrolase I ofEscherichia coli (original) (raw)
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Atomic structure of GTP cyclohydrolase I
Structure, 1995
Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as a regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohydrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system.Results The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 å resolution. The functional enzyme is a homodecameric complex with D5 symmetry, forming a torus with dimensions 65 å × 100 å. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel β-sheets of the five monomers to form a 20-stranded antiparallel β-barrel of 35 å diameter. Two pentamers are tightly associated by intercalation of two antiparallel ...
Active site topology and reaction mechanism of GTP cyclohydrolase I
Proceedings of the National Academy of Sciences, 1995
GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may ...
Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase
Acta Crystallographica Section D-biological Crystallography, 2000
DOPA decarboxylase is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of l-3,4-dihydroxyphenylalanine (l-DOPA) and l-5-hydroxytryptophan, respectively. The crystals of recombinant DOPA decarboxylase differ from those previously reported for the enzyme puri®ed from pig kidney. They belong to space group P622 with unit-cell dimensions a = b = 302.6, c = 178.1 A Ê . Both the self-rotation function and the good diffraction quality of these crystals (2.5 A Ê on a synchrotron source) suggest that there should be at least three protein dimers in the asymmetric unit. Diffraction data sets have been collected for the native enzyme and a heavy-atom derivative.
Acta Crystallographica Section B Structural Science, 1988
ten neighbouring molecules are placed in the xOz (or ~0z) orientation, the origin molecule can rotate, without hindrance, from the xOz to the ~0z equilibrium position (according to the previous criterion on the H-H distance). The high steric hindrance along the b axis explains the low contraction of the b parameter with temperature, compared with that along a (Fig. 2). Concluding remarks By careful refinements, probability distributions and steric hindrance considerations we have proved that the orientational disorder of 1-iodoadamantane can be described with a Frenkel model. The rotation between the two equilibrium positions is quasi-free. An antiferroelectric order has been pointed out similar to that locally encountered in the glassy phase of 1-cyanoadamantane. Some geometrical and energy features have been qualitatively examined to explain the lack of plastic phase in comparison to other adamantane derivatives. An extension of this study will be to investigate the behaviour of mixtures of 1-cyano-and 1-iodoadamantane. If these two compounds syncrystallize, complete thermodynamic, structure and molecular-motion analyses should allow the part played by physical parameters in a glassy crystalline phase (i.e., molecular geometry, dipolar interaction, slow molecular tumbling and particularly the local order) to be quantified. The authors are grateful to M. Muller and D. Prevost for preparing the sample. References
Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAP K ), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAP K cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods: The CHAP K domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.
Protein Science, 1999
The structure of a bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase0cyclohydrolase from Escherichia coli has been determined at 2.5 Å resolution in the absence of bound substrates and compared to the NADP-bound structure of the homologous enzyme domains from a trifunctional human synthetase enzyme. Superposition of these structures allows the identification of a highly conserved cluster of basic residues that are appropriately positioned to serve as a binding site for the poly-g-glutamyl tail of the tetrahydrofolate substrate. Modeling studies and molecular dynamic simulations of bound methylene-tetrahydrofolate and NADP shows that this binding site would allow interaction of the nicotinamide and pterin rings in the dehydrogenase active site. Comparison of these enzymes also indicates differences between their active sites that might allow the development of inhibitors specific to the bacterial target.
Acta Crystallographica Section D Biological Crystallography, 2012
Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2012). D68, 541-552 Bailey et al. • Aspartic proteinases research papers
Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli
Journal of Molecular Biology, 2006
We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn 2C in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 Å ) iodine anomalous scattering and it was refined against a native dataset up to 2.0 Å resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.
Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB
The Biochemical journal, 2017
Guanosine 5'-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5'-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5'-trip...