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, th e first enzyme in the pathway converting GDP-mannose to GDP-fucose. Th e message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient i n GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehy dratase polypeptide shares 61% identity with the enzyme from Escherich ia coli, suggesting broad evolutionary conservation. Purified recombin ant enzyme utilizes NADP(+) as a cofactor and, like its E. coli counte rpart, is inhibited by GDP-fucose, suggesting that this aspect of regu lation is also conserved. We have isolated the product of the dehydrat ase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure b y electrospray ionization-mass spectrometry and high field MMR. 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 CDP-fucos e in vitro. This unequivocally demonstrates that the epimerase and red uctase activities are on a single polypeptide. Finally, we show that t he two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.