Characterization of PglJ, a Glycosyltransferase in the N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of strains have been reported to diverge from other in steps after the biosynthesis of acetylgalactosamine-α1,3-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of . Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate -acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate -acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the genus. These studies now allow detailed characterization of the later steps in the pgl pathway in strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.