Abstract
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD+ generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.
Highlights
Polyprenol-phosphate phosphoglycosyltransferases (PGTs) are integral membrane proteins that catalyze transfer of a C1-phospho-sugar from a nucleotide diphosphate sugar to polyprenol-phosphate, forming a polyprenol-diphospho-sugar product with concomitant release of a nucleotide monophosphate (NMP)
UDP is converted to a nucleotide triphosphate (NTP), which is further converted to a stable bioluminescence signal by coupling to the luciferase/luciferin reaction components present in the assay[18,19]
Using PglC from C. jejuni as a model PGT enzyme, we have shown that the UMP-Glo assay recapitulates the radioactivity-based assay for measuring enzyme activity
Summary
Polyprenol-phosphate phosphoglycosyltransferases (PGTs) are integral membrane proteins that catalyze transfer of a C1-phospho-sugar from a nucleotide diphosphate sugar to polyprenol-phosphate, forming a polyprenol-diphospho-sugar product with concomitant release of a nucleotide monophosphate (NMP) These enzymes are often referred to as “priming” glycosyltransferases due to their essential functions in initiating biosynthesis of glycans and glycoconjugates[1,2] including glycoproteins and glycolipids[3]. Biochemical and bioinformatics studies have highlighted conserved motifs and residues crucial for catalytic activity in different PGTs4,8–11, and recently the X-ray structure of MraY has been reported, providing the first structural snapshot of a PGT12 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. The assay was used to examine WecA from T. maritima, a PGT with 11 TMHs, a dramatically different architecture from the PglC enzymes from C. jejuni and H. pullorum
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