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.

Crystal structure of the pertussis toxin-ATP complex: a molecular sensor.

Pertussis toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. The protein is a hexamer containing a catalytic subunit (S1) that is tightly associated with a pentameric cell-binding component (B-oligomer). In vitro experiments have shown that ATP and a number of detergents and phospholipids assist in activating the holotoxin by destabilizing the interaction between S1 and the B-oligomer. Similar processes may play a role in the activation of pertussis toxin in vivo. In this paper we present the crystal structure of the pertussis toxin-ATP complex and discuss the structural basis for the ATP-induced activation. In addition, we propose a physiological role for the ATP effect in the process by which the toxin enters the cytoplasm of eukaryotic cells. The key features of this proposal are that ATP binding signals the arrival of the toxin in the endoplasmic reticulum and, at the same time, triggers dissociation of the holotoxin prior to membrane translocation.