Peptidoglycan Modification by the Catalytic Domain of Streptococcus pneumoniae OatA Follows a Ping-Pong Bi-Bi Mechanism of Action

Streptococcus pneumoniae among other Gram-positive pathogens produces O-acetylated peptidoglycan using the enzyme OatA. This process occurs through the transfer of an acetyl group from a donor to the hydroxyl group of an acceptor sugar. While it has been established that this process involves the extracellular, catalytic domain of OatA (SpOatAC), mechanistic insight is still unavailable. This study examined the enzymatic characteristics of SpOatAC-catalyzed reactions through analysis of both pre-steady- and steady-state kinetics. Our findings clearly show that SpOatAC follows a ping-pong bi-bi mechanism of action involving a covalent acetyl–enzyme intermediate. The modified residue was verified to be the catalytic nucleophile, Ser438. The pH dependence of the enzyme kinetics revealed that a single ionizable group is involved, which is consistent with the participation of a His residue. Single-turnover kinetics of esterase activity demonstrated that k2 ≫ k3, revealing that the rate-limiting step for the hy...