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

FEBS Letters

Bisso, Angela¹  Sturla, Laura¹  Zanardi, Davide¹  Tonetti, Michela¹  De Flora, Antonio¹ 

[1] Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV, 1, 16132 Genova, Italy

关键词: GDP-D-mannose-4;    6-dehydratase;    L-fucose;    NADP+;    Human lymphocyte;    GMD;    GDP-D-mannose-4;    6-dehydratase;    L-GMD;    long GMD (42 kDa);    M-GMD;    medium GMD (40.2 kDa);    S-GMD;    short GMD (38.7 kDa);    GST;    glutathione transferase;    DTT;    dithiothreitol;    BSA;    bovine serum albumin;    BS3;    bis(sulfosuccinimidyl)suberate;    PBS;    phosphate-buffered saline;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(99)00982-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (K _m, K _cat) 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.

【 授权许可】

Unknown