title: Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116.
abstract: The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Zack Z
authors.initials: ZZ
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Wisconsin, 440 Henry Mall, Madison, WI, USA.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Riegert
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Goneau
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Cunningham
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Schoenhofen
authors.affiliation: Department of Biochemistry, University of Wisconsin, 440 Henry Mall, Madison, WI, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Thoden
authors.affiliation: Department of Biochemistry, University of Wisconsin, 440 Henry Mall, Madison, WI, USA.
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
authors.affiliation: National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Gilbert
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D013942:Thymine Nucleotides; C023023:lipid-linked oligosaccharides; D000217:Acyltransferases; D016695:Glycosyltransferases; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; D005947:Glucose; D005690:Galactose
keywords: Campylobacter; fucosamine; ketoisomerase; lipooligosaccharide; quinovosamine