title: Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E.coli.
abstract: N-Acetyl-D-glucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an intermediate in the biosynthesis of the enterobacterial common antigen in E.coli and some O-antigen chains in gram-negative bacteria, is formed by the transfer of GlcNAc 1-P from UDP-GlcNAc to Und-P, analogous to the reaction forming GlcNAc-P-P-dolichol (GlcNAc-P-P-Dol) in mammalian cells. Since the microsomal enzyme from animal cells exhibits a strong preference for Dol-P, which contains a saturated alpha-isoprene unit, the polyisoprenyl phosphate specificity of the homologous bacterial enzyme was characterized. The enzyme remained bound to the membrane fraction when spheroplasts, formed by lysozyme-EDTA treatment, were lysed in hypotonic buffer. GlcNAc-P-P-Und synthase (GPT) activity was elevated in a strain of E.coli bearing the rfe gene, which encodes GPT on a multicopy plasmid, and virtually absent from rfe null mutants. GPT actively utilized fully unsaturated polyprenyl phosphate (Poly-P) substrates with maximal activity seen with (C55) Und-P, but was unable to utilize (C55)Dol-P. This substrate specificity contrasts with the microsomal GPT from pig brain, which actively utilized (C55)Dol-P, but not Und-P, as substrate. GPT activity bound to particulate fractions from three strains of bacilli also exhibited a strict preference for fully unsaturated Poly-P substrates. Unexpectedly, E.coli GPT activity cofractionated with the cytosolic marker enzyme, beta-galactosidase, and not the membrane-bound enzyme, D-lactate dehydrogenase, in cells disrupted in a French pressure cell. The properties and polyisoprenyl phosphate specificity of the soluble form of GPT were identical to the activity associated with the membrane preparations obtained from spheroplasts. The evolutionary and functional significance of the use of polyisoprenyl glycosyl carrier lipids with saturated alpha-isoprene units in eukaryotes remains uncertain.
authors.affiliation: Department of Biochemistry, University of Kentucky College of Medicine, Lexington 40536, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Rush
authors.forename: P D
authors.initials: PD
authors.lastname: Rick
authors.forename: C J
authors.initials: CJ
authors.lastname: Waechter
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002793:Cholic Acids / Q000494:pharmacology; D004288:Dolichol Phosphates / Q000378:metabolism; D004755:Enterobacteriaceae / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005057:Eukaryotic Cells; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011387:Prokaryotic Cells; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002793:Cholic Acids; D004288:Dolichol Phosphates; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011106:Polyisoprenyl Phosphates; C017286:enterobacterial common antigen; C009621:undecaprenyl phosphate; D014537:Uridine Diphosphate N-Acetylglucosamine; D017855:Transferases (Other Substituted Phosphate Groups); C106504:UDP-GlcNAc-undecaprenyl phosphate N-acetylglucosaminyl 1-phosphate transferase; C083617:wecA protein, E coli; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate