A S180F substitution in D-alanine aminotransferase confers resistance to β-chloro-D-alanine in Staphylococcus aureus

β-lactam antibiotics that target the synthesis of bacterial cell wall peptidoglycan (PG) remain a cornerstone of antimicrobial chemotherapy. The activity of β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) can be enhanced by the alanine analog d-cycloserine (DCS), which blocks alanine racemase (Alr1) and d-alanine ligase (Ddl) and interferes with the supply of d-alanine for PG synthesis. Here, we report that another alanine analogue antibiotic, β-chloro-d-alanine (BCDA), also interferes with the d-alanine pathway by targeting d-alanine aminotransferase (Dat), which converts pyruvate to d-alanine. A Dat S₁₈₀F substitution, identified in a BCDA resistant MRSA mutant, resulted in reduced transaminase activity. X-ray crystallographic analysis of S. aureus Dat allowed structural modeling of the Dat-S₁₈₀F variant, which revealed an active-site loop shift that altered PLP co-factor binding. Molecular docking analysis suggests that the S₁₈₀F substitution promotes BCDA-PLP adduct dissociation via a mechanism that releases inactivated BCDA, thereby conferring resistance. These findings reveal a novel mechanism of BCDA resistance in S. aureus and the therapeutic potential of targeting Alr1 and Dat with DCS and BCDA, respectively, as part of an alternative treatment strategy for MRSA infections.