Mechanism of <i>Staphylococcus aureus</i> peptidoglycan <i>O</i>-acetyltransferase A as an <i>O</i>-acyltransferase.

Abstract

The O-acetylation of exopolysaccharides, including the essential bacterial cell wall polymer peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi of animals and fungi, peptidoglycan <i>O</i>-acetyltransferase A (OatA) is predicted to be an integral membrane protein comprised of a membrane-spanning acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located in the N- and C-terminal regions of the enzyme, respectively. The recombinant C-terminal domain (OatA_C) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatA_N) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of <i>Staphylococcus aureus</i> OatA_N, which differs markedly from that predicted in silico. We present the biochemical characterization of OatA_N as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatA_N Using in situ and in vitro assays, we characterized 35 engineered OatA variants which identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group from acetyl-CoA on the catalytic Tyr residue that is located on an extracytoplasmic loop of OatA_N Further enzymatic characterization revealed that <i>O</i>-acetyl-Tyr represents the substrate for OatA_C We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatA_N and their subsequent intramolecular transfer to OatA_C for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues.