title: Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro.
abstract: We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.
authors.affiliation: Small Molecule Drug Discovery, Genetics Institute, Inc., 424 Wilkinway, Edmonton, Alberta T6M 2H8, Canada. fsullivan@genetics.com
authors.forename: F X
authors.initials: FX
authors.lastname: Sullivan
authors.forename: R
authors.initials: R
authors.lastname: Kumar
authors.forename: R
authors.initials: R
authors.lastname: Kriz
authors.forename: M
authors.initials: M
authors.lastname: Stahl
authors.forename: G Y
authors.initials: GY
authors.lastname: Xu
authors.forename: J
authors.initials: J
authors.lastname: Rouse
authors.forename: X J
authors.initials: XJ
authors.lastname: Chang
authors.forename: A
authors.initials: A
authors.lastname: Boodhoo
authors.forename: B
authors.initials: B
authors.lastname: Potvin
authors.forename: D A
authors.initials: DA
authors.lastname: Cumming
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D006154:Guanosine Diphosphate Fucose; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase