Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose Biosynthesis in Vitro
| Autor: | Francis X. Sullivan, Ronald W. Kriz, Mark Stahl, Amechand Boodhoo, Dale A. Cumming, Guang-Yi Xu, Barry Potvin, Jason C. Rouse, Ravindra Kumar, Xiao-Jia Chang |
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| Rok vydání: | 1998 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Mannose Reductase Molecular cloning Biology Transfection medicine.disease_cause Biochemistry chemistry.chemical_compound Cricetinae Complementary DNA Guanosine Diphosphate Fucose Escherichia coli medicine Animals Humans Amino Acid Sequence Cloning Molecular Molecular Biology Hydro-Lyases Base Sequence Chinese hamster ovary cell Cell Biology Molecular biology chemistry Dehydratase Sequence Alignment |
| Zdroj: | Journal of Biological Chemistry. 273:8193-8202 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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