Abstact

GmaR was identified in Listeria monocytogenes as an essential dual-function protein that regulates the MogR-mediated repression of flagellar gene expression and catalyzes the glycosylation of the flagellar protein flagellin. To elucidate the structural and molecular basis of GmaR function, we determined the crystal structure of Bacillus cereus GmaR (bcGmaR) and investigated its glycosyltransferase and regulatory activities through structure-based biochemical and mutational analyses. Unexpectedly, bcGmaR does not interact with MogR and is unlikely to function as a MogR regulator in contrast to L. monocytogenes GmaR. Instead, bcGmaR catalyzes Mg(2+)-dependent O-linked N-acetylglucosamine (GlcNAc) transfer to B. cereus flagellin, substantially increasing its thermostability. The N-terminal glycosyltransferase (GT) domain of bcGmaR adopts a three-layer GT-A type fold with a pocket, which accommodates Mg(2+) and the sugar donor UDP-GlcNAc through the canonical DxD and C-His motifs conserved among GT-A enzymes, as well as a bcGmaR-specific phiHE motif located within a unique beta-hairpin structure. Notably, the phiHE motif is critical for catalysis, potentially by providing the catalytic base. In addition to the GT domain, the tetratricopeptide repeat domain is also required for full enzymatic activity. These findings highlight the functional divergence of GmaRs among bacterial species and underscore their unique catalytic features.