Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli.
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 Km of 0.22 +/- 0.04 mM and a specific activity of 2.3 +/- 0.2 micromol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP-L-beta-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.
Sturla
L
L
Institute of Biochemistry, University of Genova, Italy.
Bisso
A
A
Zanardi
D
D
Benatti
U
U
De Flora
A
A
Tonetti
M
M
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; D005982:Glutathione Transferase; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase