Abstract
Class D β-lactamases pose an emerging threat to the efficacy of β-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other β-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation mutagenesis at position V117 in the class D β-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (D, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO(2) binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D β-lactamases and BlaR β-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.