Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates.

Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linear polyprenols are constrained by double bond geometry and lend themselves well to interactions with polytopic membrane proteins, in which multiple transmembrane helices form a rich landscape for interactions. Recently, a new superfamily of monotopic phosphoglycosyl transferase enzymes has been identified that interacts with polyprenol phosphate substrates via a single reentrant membrane helix. Intriguingly, despite the dramatic differences in their membrane-interaction domains, both polytopic and monotopic enzymes similarly favor a unique cis/trans geometry in their polyprenol phosphate substrates. Herein, we present a multipronged biochemical and biophysical study of PglC, a monotopic phosphoglycosyl transferase that catalyzes the first membrane-committed step in N-linked glycoprotein biosynthesis in Campylobacter jejuni. We probe the significance of polyprenol phosphate geometry both in mediating substrate binding to PglC and in modulating the local membrane environment. Geometry is found to be important for binding to PglC; a conserved proline residue in the reentrant membrane helix is determined to drive polyprenol phosphate recognition and specificity. Pyrene fluorescence studies show that polyprenol phosphates at physiologically-relevant levels increase the disorder of the local lipid bilayer; however, this effect is confined to polyprenol phosphates with specific isoprene geometries. The molecular insights from this study may shed new light on the interactions of polyprenol phosphates with diverse membrane-associated proteins in glycoconjugate biosynthesis.

An operon encoding enzymes for synthesis of a putative extracellular carbohydrate attenuates acquired vancomycin resistance in Streptomyces coelicolor.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid-linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. Strains of Streptomyces coelicolor with mutations in the gene ppm1, encoding polyprenol phosphate mannose synthase, and in pmt, encoding a protein O-mannosyltransferase, are resistant to phage ϕC31 and have greatly increased susceptibility to some antibiotics, including vancomycin. In this work, second-site suppressors of the vancomycin susceptibility were isolated. The suppressor strains fell into two groups. Group 1 strains had increased resistance to vancomycin, teicoplanin and ß-lactams, and had mutations in the two-component sensor regulator system encoded by vanSR, leading to upegulation of the vanSRJKHAX cluster. Group 2 strains only had increased resistance to vancomycin and these mostly had mutations in sco2592 or sco2593, genes that are derepressed in the presence of phosphate and are likely to be required for the synthesis of a phosphate-containing extracellular polymer. In some suppressor strains the increased resistance was only observed in media with limited phosphate (mimicking the phenotype of wild-type S. coelicolor), but two strains, DT3017_R21 (ppm1-vanR-) and DT3017_R15 (ppm1- sco2593-), retained resistance on media with high phosphate content. These results support the view that vancomycin resistance in S. coelicolor is a trade-off between mechanisms that confer resistance and at least one that interferes with resistance mediated through the sco2594-sco2593-sco2592 operon.

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1- mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1- strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Stereochemical Divergence of Polyprenol Phosphate Glycosyltransferases.

In the three domains of life, lipid-linked glycans contribute to various cellular processes ranging from protein glycosylation to glycosylphosphatidylinositol anchor biosynthesis to peptidoglycan assembly. In generating many of these glycoconjugates, phosphorylated polyprenol-based lipids are charged with single sugars by polyprenol phosphate glycosyltransferases. The resultant substrates serve as glycosyltransferase donors, complementing the more common nucleoside diphosphate sugars. It had been accepted that these polyprenol phosphate glycosyltransferases acted similarly, given their considerable sequence homology. Recent findings, however, suggest that matters may not be so simple. In this Opinion we propose that the stereochemistry of sugar addition by polyprenol phosphate glycosyltransferases is not conserved across evolution, even though the GT-A fold that characterizes such enzymes is omnipresent.