A DISULFIDE BRIDGE NEAR THE ACTIVE-SITE OF CARBAPENEM-HYDROLYZING CLASS-A BETA-LACTAMASES MIGHT EXPLAIN THEIR UNUSUAL SUBSTRATE PROFILE

Bacterial resistance to beta-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of S-lactamases , enzymes that efficiently hydrolyze the amide bond of the p-lactam nu cleus. Imipenem and other carbapenems escape the activity of most acti ve site serine beta-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Thei r usefulness is, however, threatened by the appearance of new beta-lac tamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenema ses, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of beta-lactama ses. In turn, this will contribute to the design of better antibacteri al drugs. Three-dimensional models of the two class A carbapenemases w ere constructed by homology modeling. They suggested the presence, nea r the active site of the enzymes, of a disulfide bridge (C69-C238) who se existence was experimentally confirmed, Kinetic parameters were mea sured with the purified Sme-1 carbapenemase, and an attempt was made t o explain its specific substrate profile by analyzing the structures o f minimized Henri-Michaelis complexes and comparing them to those obta ined for the ''classical'' TEM-1 beta-lactamase. The peculiar substrat e profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 2 7:47-58 (C) 1997 Wiley-Liss, Inc.