المؤلفون: Cheng, Zishuo, VanPelt, Jamie, Bergstrom, Alexander, Bethel, Christopher, Katko, Andrew, Miller, Callie, Mason, Kelly, Cumming, Erin, Zhang, Huan, Kimble, Robert L, Fullington, Sarah, Bretz, Stacey Lowery, Nix, Jay C, Bonomo, Robert A, Tierney, David L, Page, Richard C, Crowder, Michael W

المصدر: Biochemistry. 57(35)

مصطلحات موضوعية: Inorganic Chemistry, Chemical Sciences, Infectious Diseases, Binding Sites, Catalytic Domain, Crystallography, X-Ray, Kinetics, Models, Molecular, Phylogeny, Protein Conformation, Spirochaeta, Zinc, beta-Lactamases, beta-Lactams, Medicinal and Biomolecular Chemistry, Biochemistry and Cell Biology, Medical Biochemistry and Metabolomics, Biochemistry & Molecular Biology, Biochemistry and cell biology, Medical biochemistry and metabolomics, Medicinal and biomolecular chemistry

الوصف: In an effort to evaluate whether a recently reported putative metallo-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MβLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MβLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of β-lactam antibiotics. This study reveals a novel metal binding site in MβLs and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.

وصف الملف: application/pdf

URL الوصول: https://escholarship.org/uc/item/0k4109qz

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المؤلفون: David L. Tierney, Lindsay Moritz, Abraham Jon Moller, Mahesh Aitha, Alex Bergstrom, Robert A. Bonomo, Amy R. Marts, Michael W. Crowder, Lucien H. Turner, Richard C. Page, Jay C. Nix

المصدر: Biochemistry

مصطلحات موضوعية: Models, Molecular, Stereochemistry, Kinetics, chemistry.chemical_element, Zinc, Crystallography, X-Ray, Biochemistry, Article, beta-Lactamases, Metal, Hydrolase, Escherichia coli, Binding site, Cloning, Molecular, chemistry.chemical_classification, Binding Sites, Spectrum Analysis, Substrate (chemistry), biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses, Up-Regulation, Enzyme, chemistry, visual_art, Pseudomonas aeruginosa, visual_art.visual_art_medium, Stoichiometry

الوصف: This study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 A, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.

URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4bf91f4b903333880ab657c88dd7a392
http://europepmc.org/articles/PMC4245990

المؤلفون: Elizabeth J. Goldsmith, Sheng Ye, Gary S. Coombs, Robert C. Bergstrom, Edwin L. Madison, David R. Corey

المصدر: Biochemistry. 42:5395-5402

مصطلحات موضوعية: Proteases, Proteolysis, medicine.medical_treatment, Biochemistry, Evolution, Molecular, Scissile bond, Catalytic Domain, medicine, Micrococcal Nuclease, Trypsin, Serine protease, chemistry.chemical_classification, Protease, biology, medicine.diagnostic_test, Hydrolysis, Serine Endopeptidases, Urokinase-Type Plasminogen Activator, Peptide Fragments, Kinetics, Enzyme, Gene Expression Regulation, chemistry, biology.protein, Thermodynamics, Plasminogen activator, Protein Binding, medicine.drug

الوصف: Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.

URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::716b9bf539431d7718584bc47a2b7edc
https://doi.org/10.1021/bi027417x

المؤلفون: Donald E. Bergstrom, Andy LiWang, Peiming Zhang, Douglas A. Klewer, Davisson Vj

المصدر: Biochemistry. 40:1518-1527

مصطلحات موضوعية: Circular dichroism, Stereochemistry, medicine.drug_class, Triazole, Carboxamide, Biochemistry, Nucleobase, chemistry.chemical_compound, Ribavirin, medicine, Phosphorus-31 NMR spectroscopy, Glycosides, Base Pairing, Nuclear Magnetic Resonance, Biomolecular, Circular Dichroism, Nucleic Acid Heteroduplexes, Temperature, Phosphorus Isotopes, Hydrogen Bonding, Sequence Analysis, DNA, Amides, Oligodeoxyribonucleotides, chemistry, Helix, Nucleic Acid Conformation, Thermodynamics, Protons, Two-dimensional nuclear magnetic resonance spectroscopy, DNA

الوصف: The concept of using a dynamic base-pairing nucleobase as a mode for degenerate recognition presents a unique challenge to analysis of DNA structure. Proton and phosphorus NMR studies are reported for two nine-residue DNA oligodeoxyribonucleotides, d(CATGGGTAC).d(GTACNCATG) (1) and d(CATGTGTAC).(GTACNCATG) (2), which contained 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (N) in the center of the helix at position 14. The duplexes were compared to the canonical Watson-Crick duplexes, d(CATGGGTAC).d(GTACCCATG) (3) and d(CATGTGTAC).d(GTACACATG) (4). Two-dimensional NOESY spectra of 1-4 in H(2)O and D(2)O solutions collected at 5 degrees C allowed assignment of the exchangeable and nonexchangeable protons for all four oligodeoxyribonucleotides. Thermodynamic and circular dichroism data indicated that 1-4 formed stable, B-form duplexes at 5 degrees C. Two-dimensional (1)H-(31)P correlation spectra indicated that there were minor perturbations in the backbone only near the site of the triazole base. Strong NOESY cross-peaks were observed between the H5 and H1' of N14 in 1 and, unexpectedly, 2, which indicated that, in both duplexes, N14 was in the syn(chi)() conformation about the glycosidic bond. NOESY spectra of 1 and 2 recorded in 95% H(2)O, 5% D(2)O indicated that the imino proton of the base opposite N14, G5, or T5, formed a weak hydrogen bond with N14. These conformations place the polar carboxamide functional group in the major groove with motional averaging on the intermediate time scale.

URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::1aab6650400b55e7ca7c753b322c6488
https://doi.org/10.1021/bi001448f

المؤلفون: Cheng, Zishuo, VanPelt, Jamie, Bergstrom, Alexander, Bethel, Christopher, Katko, Andrew, Miller, Callie, Mason, Kelly, Cumming, Erin, Zhang, Huan, Kimble, Robert L., Fullington, Sarah, Bretz, Stacey Lowery, Nix, Jay C., Bonomo, Robert A., Tierney, David L., Page, Richard C., Crowder, Michael W.

المصدر: Biochemistry; August 2018, Vol. 57 Issue: 35 p5218-5229, 12p

مستخلص: In an effort to evaluate whether a recently reported putative metallo-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinaewas overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2site that is similar to that in NDM-1 and other subclass B1 MβLs; however, the Zn1metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1metal is displaced nearly 1 Å from the position reported in other MβLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of β-lactam antibiotics. This study reveals a novel metal binding site in MβLs and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.

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المؤلفون: Aitha, Mahesh, Marts, Amy R., Bergstrom, Alex, Møller, Abraham Jon, Moritz, Lindsay, Turner, Lucien, Nix, Jay C., Bonomo, Robert A., Page, Richard C., Tierney, David L., Crowder, Michael W.

المصدر: Biochemistry; 11/25/2014, Vol. 53 Issue 46, p7321-7331, 11p

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