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Bacterial Enzyme (bacterial + enzyme)
Selected AbstractsCharacterization of carbonic anhydrase from Neisseria gonorrhoeaeFEBS JOURNAL, Issue 6 2001Björn Elleby We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source] Conformation-dependent intermolecular interaction energies of the triphosphate anion with divalent metal cations.JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 2 2004Application to the ATP-binding site of a binuclear bacterial enzyme. Abstract We have explored the conformation-dependent interaction energy of the triphosphate moiety, a key constituent of ATP and GTP, with a closed-shell divalent cation, Zn2+, used as a probe. This was done using the SIBFA polarizable molecular mechanics procedure. We have resorted to a previously developed approach in which triphosphate is built out from its elementary constitutive fragments, and the intramolecular, interfragment, interaction energies are computed simultaneously with their intermolecular interactions with the divalent cation. This approach has enabled reproduction of the values of the intermolecular interaction energies from ab initio quantum-chemistry with relative errors <3%. It was extended to the complex of a nonhydrolyzable analog of ATP with the active site of a bacterial enzyme having two Mg2+ cations as cofactors. We obtained following energy-minimization a very close overlap of the ATP analog over its position from X-ray crystallography. For models of the ATP analog,enzyme complex encompassing up to 169 atoms, the values of the SIBFA interaction energies were found to match their DFT counterparts with relative errors of <2%. © 2003 Wiley Periodicals, Inc. J Comput Chem 25: 160,168, 2004 [source] Hyperpolarized 13C magnetic resonance detection of carboxypeptidase G2 activityMAGNETIC RESONANCE IN MEDICINE, Issue 5 2009Yann Jamin Abstract Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance 13C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with 13C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc. [source] Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnelsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010A. Stsiapanava The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P212121, while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22,Å, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent. [source] X-ray structure of azide-bound fully oxidized cytochrome c oxidase from bovine heart at 2.9,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000Ming Jie Fei Two azide ions were identified, one between the Fe and Cu atoms in the O2 -reduction site and the other at the transmembrane surface of the enzyme, in the crystal structure of the azide-bound form of bovine heart cytochrome c oxidase at 2.9,Å resolution. Two geometries, a ,-1,3 type geometry between the Fe and Cu atoms and a terminal geometry on the Fe atom, are equally possible for an azide ion in the O2 -reduction site. The other azide molecule was hydrogen bonded to an amide group of an asparagine and a hydroxyl group of tyrosine in a ,-1,1 type geometry. The antisymmetric infrared bands arising from these azide ions, which show essentially identical intensity [Yoshikawa & Caughey (1992), J. Biol. Chem.267, 9757,9766], strongly suggest terminal binding of the azide to Fe. The electron density of all three imidazole ligands to CuB was clearly seen in the electron-density map of the azide-bound form of bovine heart enzyme, in contrast to the crystal structure of the azide-bound form of the bacterial enzyme [Iwata et al. (1995), Nature (London), 376, 660,669], which lacks one of the three imidazole ligands to CuB. [source] Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage,reunion domain of Escherichia coli DNA gyraseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Marcus J. Edwards Crystals of a complex formed between the 59,kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage,reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I -centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75,Å from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development. [source] Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase from Clostridium botulinum in the presence of its substrate pyruvateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Sarah C. Atkinson In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2,Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3,Å, , = 126.3°. The crystal volume per protein weight (VM) was 2.3,Å3,Da,1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Jarrod E. Voss Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba -DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba -DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba -DHDPS prepared in 0.2,M sodium fluoride, 20%(w/v) PEG 3350 and 0.1,M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15,Å are presented. The pending crystal structure of the pyruvate-bound form of Ba -DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [source] Structure-based Optimization of MurF InhibitorsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2006Geoffrey F. Stamper The d -Ala- d -Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency. We report here a multidisciplinary approach to this effort that allowed for rapid generation of cocrystal structures, thereby providing the crystallographic information critical for driving the inhibitor optimization process. This effort resulted in the discovery of low-nanomolar inhibitors of this bacterial enzyme. [source] In-vitro evaluation of khaya and albizia gums as compression coatings for drug targeting to the colonJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2005Oluwatoyin A. Odeku Khaya and albizia gums were evaluated as compression coatings for target drug delivery to the colon using indometacin (a water insoluble drug) and paracetamol (a water soluble drug) as model drugs. The core tablets were compression-coated with 300 and 400mg of 100% khaya gum, 100% albizia gum and a mixture of khaya and albizia gum (1:1). Drug release studies were carried out in 0.1M HCI (pH 1.2) for 2h, Sorensen's buffer (pH 7.4) for 3 h and then in phosphate-buffered saline (pH 6.8) or in simulated colonic fluid for the rest of the experiment to mimic the physiological conditions from the mouth to colon. The results indicated that khaya and albizia gums were capable of protecting the core tablet in the physiological environment of the stomach and small intestine, with albizia gum showing greater ability than khaya gum. The release from tablets coated with the mixture of khaya and albizia gums was midway between the two individual gums, indicating that there was no interaction between the gums. Studies carried out using rat caecal matter in phosphate-buffered saline at pH 6.8 (simulated colonic fluid) showed that the gums were susceptible to degradation by the colonic bacterial enzymes, leading to release of the drug. The results demonstrate that khaya gum and albizia gum have potential for drug targeting to the colon. [source] Structure and function of the Mur enzymes: development of novel inhibitorsMOLECULAR MICROBIOLOGY, Issue 1 2003Ahmed El Zoeiby Summary One of the biggest challenges for recent medical research is the continuous development of new antibiotics interacting with bacterial essential mechanisms. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy. The cytoplasmic steps of the biosynthesis of peptidoglycan precursor, catalysed by a series of Mur enzymes, are excellent candidates for drug development. There has been growing interest in these bacterial enzymes over the last decade. Many studies attempted to understand the detailed mechanisms and structural features of the key enzymes MurA to MurF. Only MurA is inhibited by a known antibiotic, fosfomycin. Several attempts made to develop novel inhibitors of this pathway are discussed in this review. Three novel inhibitors of MurA were identified recently. 4-Thiazolidinone compounds were designed as MurB inhibitors. Many phosphinic acid derivatives and substrate analogues were identified as inhibitors of the MurC to MurF amino acid ligases. [source] Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeriACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Xavier Carpena Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P21, with unit-cell parameters a = 60.6, b = 153.9, c = 109.2,Å, , = 102.8°. From these crystals a diffraction data set to 1.8,Å resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I212121), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5,Å. These crystals diffracted beyond 2.2,Å resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis. [source] Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Tania F. Oliveira Glutamate dehydrogenase (EC 1.4.1.2,4) from Peptoniphilus asaccharolyticus has been expressed as a selenomethionine-derivatized recombinant protein and diffraction-quality crystals have been grown that are suitable for structure determination. Preliminary structural analyses indicate that the protein assembles as a homohexameric enzyme complex in solution, similar to other bacterial and mammalian enzymes to which its sequence identity varies between 25 and 40%. The structure will provide insight into its preference for the cofactor NADH (over NADPH) by comparisons with the known structures of mammalian and bacterial enzymes. [source] Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocidaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Harkewal Singh Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomonoesters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0,Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85,Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85,Å resolution using MOSFLM and SCALA. [source] | ||||||||||||||||