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 GDPfucose, 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.Fucose is found as a component of glycoconjugates such as glycoproteins and glycolipids in a wide range of species from humans to bacteria. For example, fucose is a component of the capsular polysaccharides and antigenic determinants of bacteria, while in mammals fucose is present in many glycoconjugates, the most widely known being the human blood group antigens. Fucose-containing glycoconjugates have been implicated as playing key roles in embryonic development in the mouse (1) and more recently in the regulation of the immune response, specifically as a crucial component of the selectin ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, fucose is transferred from GDP-fucose to glycoconjugate acceptors by specific transferases. Thus, defects in GDP-fucose biosynthesis will affect all fucosylation within the cell. Recently, individuals deficient in the biosynthesis of GDP-fucose have been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II (LADII).1 These patients fail to synthesize fucosylated blood groups, and their leukocytes do not express the fucose containing carbohydrate sialyl Lewis X. The patient's leukocytes do not extravasate normally, which leads to recurrent infections.In his pioneering work in the early 1960s, Ginsberg (5, 6) elucidated the enzymatic pathway converting GDP-mannose to GDP-fucose. Later, Yurchenco and Atkinson (7) showed that this was the primary biosynthetic route to GDP-fucose. As shown in Fig. 1, GDP-mannose is converted to GDP-fucose by GDP-mannose 4,6-dehydratase via the oxidation of mannose at C-4 followed by the reduction of C-6 to a methyl group, yielding GDP-4-keto-6-deoxymannose. The reaction has been reported to proceed with transfer of a hydride fro...

Paramecium bursaria Chlorella Virus 1 Encodes Two Enzymes Involved in the Biosynthesis of GDP-L-fucose and GDP-D-rhamnose

“…The virus GMER behaves as a bifunctional enzyme, with an epimerization activity that leads to a change in the sugar configuration, followed by the NADPHdependent reduction of the 4-keto group of the intermediate to form GDP-L-fucose. These GMD and GMER activities are identical to those of GMD and GMER enzymes from prokaryotes, plants, and animals (11)(12)(13)(14)(15).…”

“…In the present study we characterize two PBCV-1 ORFs, A118R and A295L, that encode proteins that resemble GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER), respectively. GMD and GMER are highly conserved proteins involved in GDP-L-fucose formation in bacteria, plants, and animals (11)(12)(13)(14)(15). Enzyme analyses, performed on both the recombinant proteins and extracts from PBCV-1-infected Chlorella cells, indicate that both proteins are functionally active, leading to the production of two nucleotide sugars, GDP-Lfucose and GDP-D-rhamnose.…”

The GPI-linked Ly-6 Antigen E48 Regulates Expression Levels of the FX Enzyme and of E-selectin Ligands on Head and Neck Squamous Carcinoma Cells

“…The GDP-L-fucose product is the donor carbohydrate utilized by a variety of fucosyl-transferases that add it to various lactosamine acceptors, decorating both glycoproteins and glycolipids (33,34). Results by Ohyama et al (34) suggested that the transcription of FX and possibly also GDP-mannose 4,6-dehydratase is regulated by metabolites generated in the fucose synthesis pathway.…”

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

“…Biosynthesis of GDP-L-fucose is mediated by mannose 4,6-dehydratase, 3,5 epimerase and 4-ketoreductase activity in both eukaryotic and prokaryotic organism. 44,45 Accordingly, Ata12 (mannose 4,6-dehydratase) and Ata17 (epimerase/dehydratase) may be involved in the synthesis of GDP-D-mannose. Furthermore, the unsaturated hexofuranose moiety could be synthesized from GDP-D-rhamnose through Ata10 (oxidoreductase), and Ata13 (mannosyltransferase) could link it to that D-rhamnose.…”