Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro

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Abstract

We have cloned the cDNA encoding human GDP-man-nose 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.

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Sullivan, F. X., Kumar, R., Kriz, R., Stahl, M., Xu, G. Y., Rouse, J., … Cumming, D. A. (1998). Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. Journal of Biological Chemistry, 273(14), 8193–8202. https://doi.org/10.1074/jbc.273.14.8193

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