Mechanism of Staphylococcus aureus peptidoglycan O-acetyltransferase A as O-acyltransferase
Enzymes composed of transmembrane domains of acyltransferase-3 (AT-3) catalyze the O-acetylation of various extracytoplasmic glycans in all life forms. In many cases, such as peptidoglycan, these glycans are important components of the cell wall and their O-acetylation confers resistance to lytic enzymes. The enzyme responsible for O-acetylation of peptidoglycan in Gram-positive bacteria, OatA, is a single bimodal protein from an AT-3 domain fused to an SGNH domain. The AT-3 domain adopts a different topology from that predicted in silico. In addition, its use of a unique mechanism for translocation of acetyl groups across the cytoplasmic membrane for their transfer to peptidoglycan involving Tyr and Ser catalytic residues may be widely applicable to homologues involved in the modification of other important glycans of the cell membran.
The O-acetylation of exopolysaccharides, including the essential polymer of the bacterial cell wall, peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi apparatus of animals and fungi, peptidoglycan O-acetyltransferase A (OatA) should be an integral membrane protein composed of a transmembrane acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located respectively in the N- and C-terminal regions of the enzyme. The recombinant C-terminal domain (OatAVS) was characterized as an NHGS acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatANOT) or its homologues associated with other acyltransferases. We report here the experimental determination of the topology of Staphylococcus aureus OatsANOT, which differs significantly from that predicted in silico. We present the biochemical characterization of OatANOT in the context of recombinant OatA and demonstrate that acetyl-CoA serves as a substrate for OatANOT. Using in situ and in vitro assays, we characterized 35 modified OatA variants that identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group of acetyl-CoA on the catalytic Tyr residue located on an extracytoplasmic loop of OatANOT. Further enzymatic characterization revealed that O-acetyl-Tyr represents the substrate of OatAVS. We propose an action model of OatA involving the translocation of acetyl groups of acetyl-CoA across the cytoplasmic membrane by OatANOT and their subsequent intramolecular transfer to OatAVS for the O-acetylation of peptidoglycan via the concerted action of the catalytic residues Tyr and Ser.
Author contributions: research designed by CSJ and AJC; CSJ and ACA have done research; CSJ, ACA and AJC analyzed the data; and CSJ and AJC wrote the paper.
The authors declare no competing interests.
This article is a direct PNAS submission.
This article contains additional information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2103602118/-/DCSupplemental.
Raw files not processed for HerThe OatA tryptic peptide LC-MS, the corresponding Peaks XPro search results, and the annotated MS2 spectra for the support peptides have been deposited with Figshare (46??–48). All other study data is included in the main text and / or SI Annex.
- Has received July 29, 2021.
- Accepted July 23, 2021.