Abstract
The THIN-B metallo-beta-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Cephalosporins / metabolism
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Chelating Agents / pharmacology
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Chromatography, Ion Exchange
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Culture Media
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Electrophoresis, Polyacrylamide Gel
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Enzyme Inhibitors / pharmacology
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Kinetics
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Molecular Sequence Data
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Proteobacteria / enzymology*
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Proteobacteria / genetics
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Sulfhydryl Compounds / metabolism
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Trypsin / chemistry
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beta-Lactamases / genetics
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beta-Lactamases / isolation & purification
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beta-Lactamases / metabolism*
Substances
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Cephalosporins
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Chelating Agents
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Culture Media
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Enzyme Inhibitors
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Recombinant Proteins
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Sulfhydryl Compounds
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Trypsin
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beta-lactamase THIN-B
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beta-Lactamases