Structural basis for antibiotic resistance by chloramphenicol acetyltransferase type A in Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) exemplifies high-level antibiotic resistance in this major human pathogen. Its resistance to chloramphenicol is majorly conferred by enzymatic inactivation via chloramphenicol acetyltransferases (CATs). This modification sterically blocks the antibiotic's ribosomal binding and thus neutralizes its inhibitory potency. Although CATs have been structurally studied across diverse bacteria species, the structures of S. aureus CATs (saCATs) have remained uncharacterized. To address this gap and elucidate species-specific resistance mechanisms, we determined the first high-resolution crystal structure of saCAT1, the prototypical saCAT enzyme. Structural analysis delineates the active site architecture and reveals the molecular basis for substrate recognition of both chloramphenicol and fusidic acid (FA). Further enzymatic assays demonstrated that the K_m value against chloramphenicol is 16.9 µM, and the K_i value of the inhibitor FA is 83.7 µM, indicating that the inhibitory capacity of FA is relatively limited. These findings provide an essential structural framework for understanding chloramphenicol resistance in S. aureus and facilitate the rational design of novel antimicrobial strategies to combat multidrug-resistant pathogens.