title: Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase.
abstract: GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the 'de novo' pathway of GDP-L-fucose biosynthesis. The reported cDNA sequences for human GMD predict three forms of different length, whose 'in vivo' occurrence and molecular 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 recovered 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 (Km, Kcat) are apparently identical and both forms are non-competitively feedback-inhibited by GDP-L-fucose to a similar extent. M-GMD is the predominant enzyme form expressed in several human cell lines. These data seem to suggest that modulation of the 'de novo' pathway of GDP-L-fucose biosynthesis involves mechanisms other than differential 'in vivo' expression of GMD forms.
authors.affiliation: Department of Experimental Medicine, University of Genova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Bisso
authors.forename: L
authors.initials: L
authors.lastname: Sturla
authors.forename: D
authors.initials: D
authors.lastname: Zanardi
authors.forename: A
authors.initials: A
authors.lastname: De Flora
authors.forename: M
authors.initials: M
authors.lastname: Tonetti
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D009249:NADP; D005982:Glutathione Transferase; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase