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

Streptococcus pneumoniaeamong 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 SpOatACfollows 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 hydrolytic reaction was the breakdown of the acetyl–enzyme intermediate with a half-life of >1 min. The previous assignment of Asn491 as an oxyanion hole residue was also confirmed as its replacement with Ala resulted in a 50-fold decrease in catalytic efficiency relative to that of wild-type SpOatAC. However, this loss of catalytic efficiency was mostly due to a large increase in KM, suggesting that Asn491 contributes more to substrate binding.