A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase

FEMS Microbiol Lett. 2003 Aug 29;225(2):183-8. doi: 10.1016/S0378-1097(03)00448-8.

Abstract

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Base Sequence
  • Cephamycins / metabolism
  • Cephamycins / pharmacology
  • Drug Resistance, Bacterial / genetics
  • Enzyme Inhibitors / pharmacology
  • Isoelectric Point
  • Kinetics
  • Klebsiella pneumoniae / enzymology
  • Klebsiella pneumoniae / genetics*
  • Molecular Sequence Data
  • Molecular Weight
  • Plasmids*
  • Substrate Specificity
  • beta-Lactamases / classification
  • beta-Lactamases / genetics
  • beta-Lactamases / isolation & purification
  • beta-Lactamases / metabolism*

Substances

  • Anti-Bacterial Agents
  • Cephamycins
  • Enzyme Inhibitors
  • beta-Lactamases