EXPRESSION, PURIFICATION AND CHARACTERIZATION OF GDP-D-MANNOSE 4,6-DEHYDRATASE FROM ESCHERICHIA-COLI

GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathwa y that converts GDP-D-mannose to GDP-L-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and seq uenced in E. coli. Based on this sequence, we have expressed and purif ied GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then chara cterized, The catalytically active form of both enzyme species seems t o be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion pr otein has a K-m of 0.22 +/- 0.04 mM and a specific activity of 2.3 +/- 0.2 mu mol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP-L-beta-fucose, the end-product of the pathway, inhibits it specifically. The GST-GMD fusion protein contains one mole of tightly bound NADP(+) per mole of hexamer. Apparently, this NAD(+) is involved in the catalytic mechani sm of GMD. (C) 1997 Federation of European Biochemical Societies.