A D-alanine aminotransferase SF substitution confers resistance to β-chloro-D-alanine in via antibiotic inactivation.

Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of d-alanine for cell wall synthesis, the target of β-lactam antibiotics. Furthermore -dependent alanine transport controls MRSA β-lactam susceptibility in chemically defined medium (CDM) in a glucose-dependent manner. Here we report that was auxotrophic for l-alanine in CDM, and that this growth defect was rescued by glucose (or compensatory mutations), but only when the alanine racemase () and d-alanine aminotransferase () genes were functional. No role was observed for the alanine dehydrogenase 1 () and genes. As previously reported, and, to a lesser extent, mutations increased susceptibility to d-cycloserine (DCS). In contrast, only mutation increased susceptibility to β-chloro-d-alanine (BCDA), suggesting distinct targets for these alanine analogue antibiotics, which act synergistically against MRSA. Genome sequencing of a BCDA-resistant mutant identified a CT mutation in , predicted to result in a SF substitution. Expression of the operon in wild-type increased BCDA resistance. and double mutants were auxotrophic for d-alanine, indicating that Dat-SF transaminase activity is impaired, a conclusion supported by enzyme assays. Structural modeling revealed an active-site loop shift in Dat-SF that altered PLP co-factor binding. Molecular docking showed that the SF substitution promotes BCDA-PLP adduct dissociation by releasing inactivated BCDA, thereby conferring resistance. These data reveal essential roles for Alr1 and Dat during growth under nutrient-limiting conditions and the potential of combination therapy separately targeting both enzymes with DCS and BCDA to extend the treatment options for MRSA infections.