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 (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).
Alba
Jimena
J
Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438-540, Japan.
Bauvois
Cedric
C
Ishii
Yoshikazu
Y
Galleni
Moreno
M
Masuda
Katsuyoshi
K
Ishiguro
Masaji
M
Ito
Masahiko
M
Frere
Jean-Marie
JM
Yamaguchi
Keizo
K
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002513:Cephamycins / Q000378:metabolism / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D002513:Cephamycins; D004791:Enzyme Inhibitors; D001618:beta-Lactamases