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 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.