Anderson Alexander C, Schultz Bailey J, Snow Eric D, Brott Ashley S, Stangherlin Stefen, Malloch Tyler, London Jalen R, Walker Suzanne, Clarke Anthony J
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
J Biol Chem. 2025 Apr 23;301(6):108531. doi: 10.1016/j.jbc.2025.108531.
Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O-acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O-acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O-acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOAT's C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O-acetylate alginate, cellulose, and secondary cell wall polysaccharides.
细菌细胞壁聚合物通常会被酰基修饰，这些酰基赋予细菌适应性优势。许多聚合物酰化途径涉及膜结合的O-酰基转移酶（MBOAT）和SGNH家族蛋白对。例如，革兰氏阴性菌中的肽聚糖O-乙酰化需要MBOAT蛋白PatA和SGNH蛋白PatB。目前尚不清楚MBOAT-SGNH转移酶如何将酰基从细胞质中产生的酰基辅酶A供体转移到细胞外聚合物上。我们利用肽聚糖O-乙酰转移酶蛋白PatAB，探索MBOAT-SGNH蛋白对的作用机制。我们发现，MBOAT蛋白PatA催化其保守C端六肽基序中一个不变的酪氨酸残基的自乙酰化。我们还表明，PatB可以使用含有乙酰化酪氨酸的合成六肽将乙酰基转移到肽聚糖模拟物上。最后，我们报道了PatB的结构，发现它具有一些结构特征，这些特征决定了它作为O-乙酰转移酶的活性，并使其与其他SGNH酯酶和水解酶区分开来。综上所述，我们的结果支持了一种肽聚糖酰化模型，即MBOAT C端含酪氨酸的肽将酰基从MBOAT活性位点转运到SGNH活性位点，然后在该位点将酰基转移到肽聚糖上。该模型可能适用于其他含有MBOAT-SGNH蛋白对的系统，如那些对藻酸盐、纤维素和次生细胞壁多糖进行O-乙酰化的系统。
