Unraveling Xanthomonas acetyltransferase GumG: decoding catalytic promiscuity, understanding the mechanism, and enhancing enzymatic versatility

Abstract

Xanthan is a structurally complex exopolysaccharide produced by Xanthomonas campestris and one of the most extensively studied microbial biopolymers. As a sustainable alternative to petroleum-based polymers, its broader application requires precise control of polysaccharide decoration, yet the enzymatic basis of these modifications remains incompletely understood. Here, we characterise the activity and substrate scope of GumG, an AT-3 domain-containing membrane-bound acetyltransferase responsible for xanthan O-acetylation. Using mass spectrometry in combination with in vitro and in vivo assays, we show that GumG mediates non-specific acetylation of the outer mannose residue and displays pronounced substrate promiscuity. GumG also exhibits limited propionyltransferase activity, enabling the biosynthesis of hybrid acetylated-propionylated xanthan at an 8.27:1 ratio. Molecular docking and analysis of 31 xanthan variants identify a cytoplasmic substrate-binding pocket defined by Val67 and Phe71 that governs donor specificity, and an engineered GumG variant (F71L) shows enhanced propionyltransferase activity. In addition, a periplasmic His40-Trp143-Asp246-His297 motif is proposed to constitute the catalytic center. Together, these findings provide mechanistic insight into GumG multifunctionality and establish a framework for engineering xanthan derivatives with tailored physicochemical properties.