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Expression, purification and characterization of GDP‐ d ‐mannose 4,6‐dehydratase from Escherichia coli
Author(s) -
Sturla Laura,
Bisso Angela,
Zanardi Davide,
Benatti Umberto,
De Flora Antonio,
Tonetti Michela
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00762-x
Subject(s) - dehydratase , random hexamer , biochemistry , escherichia coli , mannose , fusion protein , enzyme , biology , guanosine diphosphate , microbiology and biotechnology , chemistry , gene , gtp' , recombinant dna , guanosine triphosphate
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 μ mol/h/mg. Ca 2+ and Mg 2+ 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.