Co‐conserved sequence motifs are predictive of substrate specificity in a family of monotopic phosphoglycosyl transferases

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane‐committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho‐sugar (NDP‐sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP‐sugar substrate specificity has remained elusive. In this work, we structurally characterize the C‐terminus of a prototypic monoPGT for the first time and show that 19 C‐terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co‐conserved sequence “fingerprints” that predict NDP‐sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP‐sugar substrate of a subset of enzymes in the superfamily that use UDP‐di‐N‐acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP‐sugar substrate specificity.