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The different inhibition mechanisms of OXA-1 and OXA-24 ?-lactamases are determined by the stability of active site carboxylated lysine.

ABSTRACT: The catalytic efficiency of class D ?-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D ?-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mM NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mM NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.

SUBMITTER: Che T

PROVIDER: S-EPMC3937681 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine.

Che Tao T Bethel Christopher R CR Pusztai-Carey Marianne M Bonomo Robert A RA Carey Paul R PR

The Journal of biological chemistry 20140117 9

The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectr ...[more]

PMID: 24443569

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Project description:The class D ?-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available ?-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the enzyme's mode of action and help define the mechanism of inhibition. When the crystal is maintained in HEPES buffer, the reaction is fast, shorter than the time scale of the Raman experiment. However, when the crystal holding solution contains 28% PEG 2000, the reaction is slower and can be recorded by Raman microscopy in real time; the inhibitor's Raman bands quickly disappear, transient features are seen due to an early intermediate, and, at approximately 2-11 min, new bands appear that are assigned to the late intermediate species. At about 50 min, bands due to all intermediates are replaced by Raman signals of the unreacted inhibitor. The new population remains unchanged indicating (i) that the OXA-24 is no longer active and (ii) that the decarboxylation of Lys84 occurred during the first reaction cycle. Using absorbance spectroscopy, a one-cycle reaction could be carried out in aqueous solution producing inactive OXA-24 as assayed by the chromogenic substrate nitrocefin. However, activity could be restored by reacting aqueous OXA-24 with a large excess of NaHCO(3), which recarboxylates Lys84. In contrast, the addition of NaHCO(3) was not successful in reactivating OXA-24 in the crystalline state; this is ascribed to the inability to create a concentration of NaHCO(3) in large excess over the OXA-24 that is present in the crystal. The finding that inhibitor compounds can inactivate a class D enzyme by promoting decarboxylation of an active site lysine suggests a novel function that could be exploited in inhibitor design.

Dataset Information

The different inhibition mechanisms of OXA-1 and OXA-24 ?-lactamases are determined by the stability of active site carboxylated lysine.

Publications

The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine.

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