Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro.
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.
Sullivan
F X
FX
Small Molecule Drug Discovery, Genetics Institute, Inc., 424 Wilkinway, Edmonton, Alberta T6M 2H8, Canada. fsullivan@genetics.com
Kumar
R
R
Kriz
R
R
Stahl
M
M
Xu
G Y
GY
Rouse
J
J
Chang
X J
XJ
Boodhoo
A
A
Potvin
B
B
Cumming
D A
DA
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
D018076:DNA, Complementary; D006154:Guanosine Diphosphate Fucose; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase