Conservation and Covariance in Small Bacterial Phosphoglycosyltransferases Identify the Functional Catalytic Core

Abstract

Phosphoglycosyltransferases (PGTs) catalyze the transfer of a C1′-phosphosugar from a soluble sugar nucleotide diphosphate to a polyprenol-phosphate. These enzymes act at the membrane interface, forming the first membrane-associated intermediates in the biosynthesis of cell-surface glycans and glycoconjugates including glycoproteins, glycolipids and the peptidoglycan in bacteria. PGTs vary greatly in both in their membrane topologies and their substrate preferences. PGTs, such as MraY and WecA, are polytopic, while other families of uniquely prokaryotic enzymes have only a single predicted transmembrane helix. PglC, a PGT involved in the biosynthesis of N-linked glycoproteins in the enteropathogen C. jejuni, is representative of one of the structurally most simple members of the diverse family of small bacterial PGT enzymes. Herein, we apply bioinformatics and covariance-weighted distance constraints in geometry- and homology-based model building, together with mutational analysis to investigate monotopic PGTs. The pool of 15,000 sequences that are analyzed include the PglC-like enzymes, as well as sequences from two other related PGTs that contain a “PglC-like” domain embedded in their larger structures (namely, the bifunctional PglB family, typified by PglB from N. gonorrheae and WbaP-like enzymes, typified by WbaP from S. enterica). Including these two sub-families of PGTs in the analysis highlights key residues conserved across all three families of small bacterial PGTs. Mutagenesis analysis of these conserved residues provides further information on the essentiality of many of these residues in catalysis. Construction of a structural model of the cytosolic globular domain utilizing three-dimensional distance constraints, provided by conservation covariance analysis, provides additional insight into the catalytic core of these families of small bacterial PGT enzymes.

A Modular Approach to Phosphoglycosyltransferase Inhibitors Inspired by Nucleoside Antibiotics

“…Furthermore, the complexity of the natural product structures makes it very challenging to repurpose the structures of the natural products, by synthesis [14][15][16] or semisynthesis, [17][18][19][20][21][22] to target alternative PGTsw ith different substrate specificities.T his challenge is furthere xacerbated when working with PGTs belongingt os tructural classes other than the wellstudied MraY and WecA-type integral membrane proteins, which feature 10 and 11 predicted TMHs, respectively.F or example,r ecent bioinformaticsa nd biochemical analysish as revealed thousands of homologous small bacterial PGTsw ith only as ingle TMH and as oluble globulard omain within a2 0-25 kDa protein. [23] While these PGTsc atalyze comparable biochemicalp rocesses and also play important roles at the initiation of diverse glycoconjugate biosynthetic pathways,t here are currently no small molecule inhibitors that can be used to inform on the biology and essentiality of particularp athways and which may ultimately representn ovel targets for therapeutic intervention.…”

Bacterial polysaccharide synthesis and export

“…Other PNPT enzymes, which utilise simple UDP-N-acetylglucosamine, exemplified by WecA, are predicted to have a periplasmic Nterminus and cytoplasmic C-terminus and consequently an odd number of helices [14,15] (Figure 2b). The PHPT class of enzymes show significant variation N-terminal to the conserved catalytically active C-terminal domain [16,17,18 ]. The most common form of PHPT, exemplified by WbaP, has a cytoplasmic N-terminus four large transmembrane helices and two shorter helices, six transmembrane helices in total, whilst PglC, which initiates the biosynthesis of the glycan precursor required for Nlinked glycosylation in Campylobacter jejuni, has a periplasmic N-terminus and a single transmembrane helix [19] (Figure 2b).…”

“…The most common form of PHPT, exemplified by WbaP, has a cytoplasmic N-terminus four large transmembrane helices and two shorter helices, six transmembrane helices in total, whilst PglC, which initiates the biosynthesis of the glycan precursor required for Nlinked glycosylation in Campylobacter jejuni, has a periplasmic N-terminus and a single transmembrane helix [19] (Figure 2b). A molecular model of the PglC catalytic domain has been generated (EV-fold and I-TASSER) and validated by site directed mutagenesis [18 ]. Potent inhibitors of PglC have also been reported [20 ].…”

A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

“…To date, biochemical and bioinformatics studies have highlighted conserved motifs and residues crucial for catalytic activity in different PGTs 4 8 9 10 11 , and recently the X-ray structure of MraY has been reported, providing the first structural snapshot of a PGT 12 . However, despite their centrality in glycoconjugate biosynthesis, the characterization of PGTs remains a challenge, due to difficulties associated with overexpressing and purifying integral membrane proteins, and because of limited access to robust assays that can be applied generally to members of the diverse family of enzymes.…”