Structure and Function of GDP-Mannose-3',5'-Epimerase: An Enzyme which Performs Three Chemical Reactions at the Same Active Site
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- 作者：Louise L. Major ; Beata A. Wolucka ; and James H. Naismith
- 刊名：Journal of the American Chemical Society
- 出版年：2005
- 出版时间：December 28, 2005
- 年：2005
- 卷：127
- 期：51
- 页码：18309 - 18320
- 全文大小：574K
- 年卷期：v.127,no.51(December 28, 2005)
- ISSN：1520-5126
文摘
GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization ofboth the 3' and 5' positions of GDP-
-D-mannose to yield GDP-
-L-galactose. Production of the C5' epimerof GDP--D-mannose, GDP--L-gulose, has also been reported. The reaction occurs as part of vitamin Cbiosynthesis in plants. We have determined structures of complexes of GME with GDP--D-mannose, GDP--L-galactose, and a mixture of GDP--L-gulose with GDP--L-4-keto-gulose to resolutions varying from2.0 to 1.4 Å. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. Wehave confirmed that GME establishes an equilibrium between two products, GDP--L-galactose and GDP--L-gulose. The reaction proceeds by C4' oxidation of GDP--D-mannose followed by epimerization of theC5' position to give GDP--L-4-keto-gulose. This intermediate is either reduced to give GDP--L-gulose orthe C3' position is epimerized to give GDP--L-4-keto-galactose, then C4' is reduced to GDP--L-galactose.The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structuralanalysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsiblefor both epimerizations. On the basis of the structure of the GDP--L-gulose/GDP--L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate islikely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract aprotein to the keto function of a carbohydrate identifies key common features.
-D-mannose to yield GDP--L-galactose. Production of the C5' epimerof GDP--D-mannose, GDP--L-gulose, has also been reported. The reaction occurs as part of vitamin Cbiosynthesis in plants. We have determined structures of complexes of GME with GDP--D-mannose, GDP--L-galactose, and a mixture of GDP--L-gulose with GDP--L-4-keto-gulose to resolutions varying from2.0 to 1.4 Å. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. Wehave confirmed that GME establishes an equilibrium between two products, GDP--L-galactose and GDP--L-gulose. The reaction proceeds by C4' oxidation of GDP--D-mannose followed by epimerization of theC5' position to give GDP--L-4-keto-gulose. This intermediate is either reduced to give GDP--L-gulose orthe C3' position is epimerized to give GDP--L-4-keto-galactose, then C4' is reduced to GDP--L-galactose.The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structuralanalysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsiblefor both epimerizations. On the basis of the structure of the GDP--L-gulose/GDP--L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate islikely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract aprotein to the keto function of a carbohydrate identifies key common features.