Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase

GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the 'de novo' path way of GDP-L-fucose biosynthesis. Th; reported cDNA sequences for human GMD predict three forms of different length, whose 'in vivo' occurrence and mo lecular properties are completely undefined, Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form . Only the 42 kDa long GMD (L-GMD) and the 40.2 kDa (M-GMD) forms were reco vered as soluble functional proteins, while the 38.7 kDa form, short GMD (S -GMD), lacking an N-terminal domain critical for dinucleotide binding, was inactive and formed a precipitate. Both L-GMD and M-GMD are homodimers and contain 1 mol of tightly bound NADP(+). Their kinetic properties (K-m, K-ca t) are apparently identical and both forms are non-competitively feedback-i nhibited by GDP-L-fucose to a similar extent, M-GMD is the predominant enzy me form expressed in several human cell lines. These data seem to suggest t hat modulation of the 'de novo' pathway of GDP-L-fucose biosynthesis involv es mechanisms other than differential 'in vivo' expression of GMD forms. (C ) 1999 Federation of European Biochemical Societies.