Mechanism of peptidoglycan -acetyltransferase A as an -acyltransferase.

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 -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) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatA) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of OatA, which differs markedly from that predicted in silico. We present the biochemical characterization of OatA as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatA 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 Further enzymatic characterization revealed that -acetyl-Tyr represents the substrate for OatA We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatA and their subsequent intramolecular transfer to OatA for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues.