Dataset Information

Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme.

ABSTRACT: Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate but is impaired with respect to deacylation. We employed the fluorescent penicillin BOCILLIN FL to show that three different substitutions for K70 (alanine, aspartate, and glutamate) lead to the accumulation of significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL deacylation rates (t(1/2)) vary depending on the identity of the substituting residue, from approximately 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e., nonfluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

SUBMITTER: Schneider KD

PROVIDER: S-EPMC2756174 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme.

Schneider Kyle D KD Bethel Christopher R CR Distler Anne M AM Hujer Andrea M AM Bonomo Robert A RA Leonard David A DA

Biochemistry 20090701 26

Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric c ...[more]

PMID: 19485421

Similar Datasets

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

Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme.

Publications

Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets