Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are kno wn, are the primary cause of resistance to beta-lactam antibiotics. The cla ss A beta-lactamases form the largest group. A novel class A beta-lactamase , named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included c arbapenem antibiotics. This is a serious development, since carbapenems hav e been relatively immune to the action of these resistance enzymes. Inhibit ors for this enzyme are sought. We describe herein that a type of monobacta m molecule of our design inactivates the NMC-A beta-lactamase rapidly, effi ciently, and irreversibly, The mechanism of inactivation was investigated b y solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 Angstro m resolution. The structure shed light on the nature of the fragmentation o f the inhibitor on enzyme acylation and indicated that there are two acyl-e nzyme species that account for enzyme inhibition. Each of these inhibited e nzyme species is trapped in a distinct local energy minimum that does not p redispose the inhibitor species for deacylation, accounting for the irrever sible mode of enzyme inhibition. Molecular dynamics simulations provided ev idence in favor of a dynamic motion for the acyl-enzyme species, which samp les a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima, A discussion of the likelihood of such dynamic motion for turnover of substrates during the nor mal catalytic processes of the enzyme is presented.