Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface

Abstract

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.

Characterization of the Exopolysaccharide Biosynthesis Pathway in Myxococcus xanthus

“…PHPTs generally utilize UDP-glucose (UDP-Glc) or UDP-galactose (UDP-Gal) as substrates to transfer Glc-1-P or Gal-1-P, respectively, to Und-P ( 29 , 61 ). Therefore, by following the same strategy as that previously reported ( 37 , 41 , 62 ), we tested whether EpsZ could functionally replace WcaJ Ec or WbaP Se , which catalyze the transfer of Glc-1-P and Gal-1-P to Und-P, respectively.…”

A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

“…In OTase-dependent glycosylation pathways, the lipid-linked glycan precursor is assembled on the cytoplasmic side of the membrane, translocated across the membrane, and then transferred onto acceptor proteins at the periplasmic side (22). We therefore hypothesized that at least four genes encoding a phosphoglycosyl transferase (initiating enzyme that mediates the synthesis of a lipidlinked sugar) (23,24), two additional glycosyltransferases, and a flippase protein would be required for the synthesis and translocation of a B. cenocepacia lipid-linked trisaccharide glycan (22,24). The chromosome 1 of B. cenocepacia has a putative five-gene operon flanked by the O-antigen synthesis cluster and lipid A-core biosynthesis genes (25) (Fig.…”

“…Predicted function of the remaining genes in the ogc cluster. To gain clues on the function of the remaining ogc genes we investigated the glycosylation status of the DsbA1 derived peptide 23 VQTSVPADSAPAASAAAAPAGLVEGQN YTVLANPIPQQQAGK 64 in the various ogc gene deletion mutants. MS spectra of the DsbA1 derived peptide in K56-2, ΔpglL, ∆ogc, ∆ogcX, ∆ogcI, ∆ogcAB and ∆ogcE are shown in Fig.…”

Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

“…PGTs are integral membrane proteins that initiate a wide variety of essential glycoconjugate biosynthesis pathways, including peptidoglycan and N-linked glycan biosynthesis, by catalyzing the transfer of a phosphosugar from a sugar nucleoside diphosphate donor to a membrane-resident polyprenol phosphate. PGTs can be grouped broadly into two superfamilies based on their membrane topology ( Lukose et al, 2017 ). One superfamily, exemplified by bacterial MraY and WecA, is composed of polytopic PGTs with 10- and 11- transmembrane helices respectively and active sites crafted from extended cytoplasmic inter-TM loops ( Anderson et al, 2000 ).…”

“…The superfamily also includes WbaP, which features a PglC-like core elaborated with four N-terminal transmembrane helices that are not necessary for catalytic activity ( Saldías et al, 2008 ), and the bifunctional enzyme PglB from Neisseria gonorrhoeae , which has an additional C-terminal aminotransferase domain ( Hartley et al, 2011 ). Topology predictions using multiple algorithms suggested that the N-terminal hydrophobic domain of PglC, and the analogous domain in other superfamily members, forms a transmembrane helix, such that the N-terminus is in the periplasm and the globular domain in the cytoplasm ( Furlong et al, 2015 ; Lukose et al, 2017 ). However, biochemical analysis of WcaJ, a WbaP homolog, supported a model wherein both termini of the corresponding domain of WcaJ are in the cytoplasm, forming a reentrant topology, rather than a membrane-spanning one ( Furlong et al, 2015 ).…”