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

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 (38 246), the N-terminal sequence (GEASPVDPLRPVV), and p/ (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 muM). 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 muM). (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.