Protein β-O-glucosylation by Legionella LtpM through short consensus sequons G-T/S and S-G.

Unlike N-glycosylation, protein O-glycosylation often lacks a strict consensus sequon, making synthesis of homogeneous O-glycoproteins and site-specific engineering of O-glucosylation challenging. Here we identify Legionella effector LtpM as a versatile protein β-O-glucosyltransferase recognizing extremely short two-residue sequons G-T/S and S-G. X-ray crystallography, molecular simulation and biochemical studies together reveal a unique catalytic mechanism: four residues of LtpM (F166, Q167, W228 and K225) serve as 'gatekeepers' above the binding pocket of the uridine diphosphate (UDP)-glucose sugar donor to form a narrow clamp for the substrate proteins, limiting the residue adjacent to serine or threonine to be exclusively glycine. By exploiting the short sequons, LtpM is developed as a powerful tool for site-specifically O-glucosylating various eukaryotic proteins of interest. In particular, O-glucose serves as a functional surrogate for O-linked N-acetylglucosamine in a synaptic Ras GTPase-activating protein. Additionally, LtpM accepts 6-azido analog of UDP-glucose and enables site-specific bioorthogonal conjugation of proteins.