Mutation of the Active Site Carboxy-Lysine (K70) of OXA-1 β-Lactamase Results in a Deacylation-Deficient Enzyme

Abstract

Class D β-lactamases hydrolyze β-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate, and subsequently employ water to deacylate the β-lactam and release product. Class D β-lactamases are carboxylated on the ε-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 β-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 for deacylation. We employed the fluorescent penicillin BOCILLIN FL™ to show that three different substitutions for K70 (alanine, aspartate and glutamate) accumulate significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL™ deacylation rates vary depending on the identity of the substituting residue, from t1/2 ≈ 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. non-fluorescent) 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.

Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii

“…13 Although these crystals yielded protein structures at high resolution, all attempts to capture ligands bound as acylintermediates failed. We then introduced mutations that are known to diminish or eliminate deacylation of substrates, 33, 34 and have been used successfully in the past to capture acyl-intermediates in class D enzymes. 7, 17 We used site-directed mutagenesis to introduce a V130D substitution into the bla OXA-160 gene and used it to express and purify the variant enzyme.…”

Site-Saturation Mutagenesis of Position V117 in OXA-1 β-Lactamase: Effect of Side Chain Polarity on Enzyme Carboxylation and Substrate Turnover

“…While quantitation of the fluorescence signal over time allowed determination of exponential deacylation rates, this assay suffered from low sensitivity and a lack of time-resolution. Deacylation could also be measured by following the quench of OXA-1 tryptophan fluorescence by the dye ligand Cibacron Blue 3GA as the hydrolyzed β-lactam product left the active site (10). This assay greatly increased the time-resolution for measuring deacylation rates, but was dependent on the unusually high environmental-sensitivity of the class D β-lactamase’s tryptophan fluorescence to Cibacron Blue.…”

Three Decades of β-Lactamase Inhibitors

“…Class D. Our understanding of the hydrolytic mechanism of class D ␤-lactamases is based on the careful study of OXA-10, -13, and -1 (253,310,334,379,398,399). This class of enzymes is unique because of the direct role of carboxylation of the active-site Lys70.…”