Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35

Antimicrob Agents Chemother. 2016 Mar 25;60(4):2366-72. doi: 10.1128/AAC.00531-15. Print 2016 Apr.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Anti-Bacterial Agents / chemistry*
  • Anti-Bacterial Agents / pharmacology
  • Biocatalysis
  • Catalytic Domain
  • Cephalosporins / chemistry*
  • Cephalosporins / pharmacology
  • Cloning, Molecular
  • Escherichia coli / drug effects*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression
  • Isoleucine / chemistry*
  • Isoleucine / metabolism
  • Kinetics
  • Models, Molecular
  • Mutation
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Serine / chemistry
  • Serine / metabolism
  • Threonine / chemistry
  • Threonine / metabolism
  • beta-Lactam Resistance / genetics*
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism

Substances

  • Anti-Bacterial Agents
  • Cephalosporins
  • Recombinant Proteins
  • Isoleucine
  • Threonine
  • loracarbef
  • Serine
  • beta-Lactamases
  • beta-lactamase NDM-1