Expression, purification and characterization of GDP‐d‐mannose 4,6‐dehydratase from Escherichia coli

FEBS Letters

Bisso, Angela¹  Benatti, Umberto¹  Sturla, Laura¹  Zanardi, Davide¹  Tonetti, Michela¹  De Flora, Antonio¹ 

[1] Institute of Biochemistry, University of Genova, Viale Benedetto XV, 1, 16132 Genova, Italy

关键词: GDP-d-mannose dehydratase;    GDP-l-fucose biosynthesis;    l-Fucose metabolism;    NADP+;    (Escherichia coli);    GDP;    guanosine diphosphate;    GST;    glutathione-S-transferase;    GMD;    GDP-d-mannose 4;    6-dehydratase;    TDP;    thymidine diphosphate;    PCR;    polymerase chain reaction;    LB broth;    Luria-Bertani broth;    IPTG;    isopropyl-β-d-thiogalactopyranoside;    PMSF;    phenylmethylsulfonyl fluoride;    EDTA;    ethylenediaminetetraacetic acid;   

DOI  :  10.1016/S0014-5793(97)00762-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

GDP-d-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-d-mannose to GDP-l-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion protein has a K _m of 0.22±0.04 mM and a specific activity of 2.3±0.2 μmol/h/mg. Ca²⁺ and Mg²⁺ activate GMD, while GDP-l-β-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 NADP⁺ is involved in the catalytic mechanism of GMD.

【 授权许可】

Unknown