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

Entova, Sonya; Guan, Ziqiang; Imperiali, Barbara

doi:10.1016/j.abb.2019.108111

Cited by 14 publications

(18 citation statements)

References 68 publications

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“…11,12,25,30 It has also recently been suggested that the unique geometry of BP may aid in binding to PGTs and may also facilitate interactions at the membrane interface. 31 It is possible that lipophilic polyisoprenoids, when added with surfactants or organic alcohols, do not necessarily share these same properties as an artifact of exogenous supplementation.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases

et al. 2019

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The protective surfaces of bacteria are comprised of polysaccharides and are involved in host invasion and colonization, host immune system evasion, as well as antibacterial resistance. A major barrier to our fundamental understanding of these complex surface polysaccharides lies in the tremendous diversity in glycan composition among bacterial species. The polyisoprenoid bactoprenyl phosphate (or undecaprenyl phosphate) is an essential lipid carrier necessary for early stages of glycopolymer assembly. Because of the ubiquity of bactoprenyl phosphate in these critical processes, molecular probes appended to this lipid carrier simplify identification of enzymatic roles during polysaccharide bioassembly. A limited number of these probes exist in the literature or have been assessed with such pathways, and the limits of their use are not currently known. Herein, we devise an efficient method for producing fluorescently modified bactoprenyl probes. We further expand our previous efforts utilizing 2-nitrileaniline, and additionally prepare nitrobenzoxadizol tagged bactoprenyl phosphate for the first time. We then assess enzyme promiscuity of these two probes utilizing four well characterized initiating phosphoglycosyltransferases: CPS2E (Streptococcus pneumoniae), WbaP (Salmonella enterica), WecA (Escherichia coli) and WecP (Aeromonas hydrophilia). Both probes serve as substrates for these enzymes and could be readily used to investigate a wide range of bacterial glycoassembly pathways. Interestingly, we have also identified unique solubility requirements for the

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Section: ■ Results and Discussionmentioning

confidence: 99%

General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases

et al. 2019

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“…The prevalence of sugar-modifying and GT bifunctional enzymes is consistent with the existence of protein-protein interactions facilitating product transfers within the respective pathways. Fusions with regulatory domains point to possible regulation of glycan biosynthesis at the PGT reaction specifically, allowing for conservative use of the limited supply of PrenP (48), the common substrate in these glycoconjugate biosynthesis pathways.…”

Section: Discussionmentioning

confidence: 99%

“…Rather, it is possible that these polyPGT F -monoPGT NF fusion enzymes resemble an ancestral enzyme. Notably, the monoPGT-core fold, exemplified by C. jejuni PglC, has been shown to increase the local membrane concentration of the PrenP substrate (48). Such an enrichment could increase catalytic throughput of the fused polyPGT enzyme and confer a selective advantage.…”

Section: Discussionmentioning

confidence: 99%

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases

O’Toole

Imperiali

Allen

2021

Proc. Natl. Acad. Sci. U.S.A.

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The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

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“…Phosphoglycosyl transferase PglC from Campylobacter consicus is an example of the other family, dual domain phosphoglycosyl transferases. Mechanistic studies on the enzyme have shown that the reaction is a two-step process with a covalent intermediate [112,113]. A nucleophilic side chain attacks on the phosphate, forming an intermediate that is attacked by the prenyl phosphate in the second step.…”

Section: Catalysis Phosphoglycosyl Transferasesmentioning

confidence: 99%

“…An aspartate in a conserved Asp-Glu dyad has been proposed as the attacking nucleophile, while the glutamic acid residue has been speculated to assist the release of UMP and nucleophilic attack by Pren-P. Phosphoglycosyl transferase PglC from Campylobacter consicus is an example of the other family, dual domain phosphoglycosyl transferases. Mechanistic studies on the enzyme have shown that the reaction is a two-step process with a covalent intermediate [112,113]. A nucleophilic side chain attacks on the phosphate, forming an intermediate that is attacked by the prenyl phosphate in the second step.…”

Section: Catalysis Phosphoglycosyl Transferasesmentioning

confidence: 99%

Nucleotide Sugars in Chemistry and Biology

Mikkola

2020

Molecules

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Nucleotide sugars have essential roles in every living creature. They are the building blocks of the biosynthesis of carbohydrates and their conjugates. They are involved in processes that are targets for drug development, and their analogs are potential inhibitors of these processes. Drug development requires efficient methods for the synthesis of oligosaccharides and nucleotide sugar building blocks as well as of modified structures as potential inhibitors. It requires also understanding the details of biological and chemical processes as well as the reactivity and reactions under different conditions. This article addresses all these issues by giving a broad overview on nucleotide sugars in biological and chemical reactions. As the background for the topic, glycosylation reactions in mammalian and bacterial cells are briefly discussed. In the following sections, structures and biosynthetic routes for nucleotide sugars, as well as the mechanisms of action of nucleotide sugar-utilizing enzymes, are discussed. Chemical topics include the reactivity and chemical synthesis methods. Finally, the enzymatic in vitro synthesis of nucleotide sugars and the utilization of enzyme cascades in the synthesis of nucleotide sugars and oligosaccharides are briefly discussed.

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

Cited by 14 publications

References 68 publications

General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases

General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases

Nucleotide Sugars in Chemistry and Biology

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