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

The SGNH hydrolase family: a template for carbohydrate diversity

“…In this pathway, an acetyl group from an acetyl coenzyme A donor is transported across the cytoplasmic membrane by the action of the N-terminal integral membrane domain, which belongs to the Acyl_Transf_3 (AT3; PF01757) family. The N-terminal domain of OatA then directly acetylates the C-terminal domain, which belongs to the SGNH-AT3 family and is also discussed here ( Jones et al. 2021 ).…”

A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer

“…The Salmonella O-antigen acetyltransferases OafB and OafA are both AT3-SGNH fusion proteins and predicted to have an 11th TMH to allow the fused SGNH domain to be located in the periplasm ( Kintz et al, 2015 ; Pearson et al, 2020 ). In contrast, Jones et al, 2021 proposed that OatA contains only 9 TMH with one large re-entrant loop; as an AT3-SGNH protein, OatA was also previously predicted to contain 11 TMH, orientating the SGNH domain in the periplasm ( Bernard et al, 2011 ; Bera et al, 2005 ). In addition, a large cytoplasmic loop was described between TMH 8 and 9 of OatA, where Oac has only short cytoplasmic loops but a longer periplasmic loop between TMH3 and 4 ( Thanweer et al, 2008 ; Rajput and Verma, 2022 ).…”

“…While the majority of AT3 domains consist of only an AT3 domain (standalone AT3), many are AT3 domains with an additional TMH and C-terminal, periplasmic SGNH domain attached via a periplasmic linking region (AT3-SGNH) ( Meziane-Cherif et al, 2015 ). Thanweer et al, 2008 and Jones et al, 2021 have performed PhoA-LacZɑ fusion analysis of the O-antigen acetyltransferase Oac from Shigella flexneri (a standalone AT3) and OatA from S. aureus (an AT3-SGNH), respectively. Analysis by Thanweer et al, 2008 suggested that Oac has 10 TMH, with the N- and C-termini in the cytoplasm.…”

“…Previous in vitro studies suggest that standalone AT3 proteins CmmA, MdmB, and Asm19 are able to utilise acetyl-CoA as an acetyl donor ( Hara and Hutchinson, 1992 ; Moss et al, 2002 ; García et al, 2011 ). Importantly, Jones et al, 2021 found OatA from S. aureus (an AT3-SGNH protein) are able to hydrolyse the acetyl group from acetyl-CoA and transfer it to the peptidoglycan-like acceptor substrate, effectively observing the entire catalytic cycle in vitro ( Jones et al, 2021 ). Thus, the available data indicate that acetyl-CoA is the most likely acetyl group donor, but how this interacts with the AT3 domain or how the acetyl group can cross the membrane, is as yet unknown.…”

“…In addition, site-directed mutagenesis identified three conserved tyrosine residues which are required for function ( Jones et al, 2021 ). One of these Tyr residues is predicted to be located in the periplasm and is proposed to be involved in transfer of the acyl group from the AT3 domain to the SGNH domain in AT3-SGNH proteins ( Jones et al, 2021 ). Furthermore, it is known that the catalytic triad of the SGNH domain of OafB from Salmonella enterica subsp.…”