Abstract
Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution
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Anti-Bacterial Agents / chemistry*
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Anti-Bacterial Agents / pharmacology
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Biocatalysis
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Catalytic Domain
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Cephalosporins / chemistry*
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Cephalosporins / pharmacology
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Cloning, Molecular
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Escherichia coli / drug effects*
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Gene Expression
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Isoleucine / chemistry*
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Isoleucine / metabolism
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Kinetics
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Models, Molecular
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Mutation
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Protein Structure, Secondary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Serine / chemistry
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Serine / metabolism
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Threonine / chemistry
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Threonine / metabolism
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beta-Lactam Resistance / genetics*
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beta-Lactamases / chemistry*
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beta-Lactamases / genetics
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beta-Lactamases / metabolism
Substances
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Anti-Bacterial Agents
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Cephalosporins
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Recombinant Proteins
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Isoleucine
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Threonine
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loracarbef
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Serine
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beta-Lactamases
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beta-lactamase NDM-1