| Bacterial resistance to current beta-lactam antibiotics represents an increasing clinical threat today. The beta-lactamase enzymes play a major role in bacterial defense mechanisms, being able to efficiently catalyze hydrolysis of the beta-lactam ring, inactivating the antibiotic. beta-lactamase inhibitors can therefore be useful agents in combating resistance. In this work we show that the class A TEM-2, class C P99 and class D OXA-1 beta-lactamases are inactivated by O-aryloxycarbonyl hydroxamates, such as N-(benzyloxycarbonyl)-O-(phenoxycarbonyl) hydroxylamine. Inhibition of the class A TEM-2 and class C P99 beta-lactamases has been shown to proceed by acylation of the active site, and result in irreversibly modified enzymes. Both N-(benzyloxycarbonyl) hydroxamic acid and phenol are displaced from the enzyme during the inactivation. A crystallographic structure of the inactivated P99 beta-lactamase showed the active site serine 64 and lysine 315 cross-linked. The O-aryloxycarbonyl hydroxamates functioned as inhibitory substrates of the class D OXA-1 beta-lactamase, with low micromolar K m's. The Actinomadura R39 DD-peptidase was also irreversibly inhibited by these molecules.;It was found that O-alkyloxycarbonyl hydroxamates bearing substituted lactic acid leaving groups undergo spontaneous rearrangement in aqueous solution to alpha-hydroxy esters, accompanied by the release of CO2. The resulting alpha-hydroxy esters were substrates of the Class C and A enzymes having kcat/Km values of up to 105 and 104 M-1 s-1, respectively. The alpha-hydroxyl substituent was shown to be crucial for interaction with the active site. Introduction of a phosphonate moiety into the reaction center of such a molecule created a micromolar competitive inhibitor of the P99 beta-lactamase. A slow, time-dependent inactivation of the enzyme was also observed, presumably reflecting phosphonylation of the active site serine. Finally, an alpha-hydroxy substituted depsipeptide was synthesized to examine the interaction of the hydroxyl in the context of a classic amido side chain. It was found that this molecule was a substrate of all three classes of the serine beta-lactamases, and two LMW DD-peptidases, from Streptomyces R61 and Actinomadura R39. The stereo-preferences at the alpha-carbon were examined and a preference for the D isomer by all the above enzymes was observed. |
