GDP-mannose-3',5'-epimerase (GME) from
Arabidopsis thaliana catalyzes the epimerization ofboth the 3' and 5' positions of GDP-
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D-mannose to yield GDP-
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L-galactose. Production of the C5' epimerof GDP-
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D-mannose, GDP-
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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-
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D-mannose, GDP-
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L-galactose, and a mixture of GDP-
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L-gulose with GDP-
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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-
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L-galactose and GDP-
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L-gulose. The reaction proceeds by C4' oxidation of GDP-
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D-mannose followed by epimerization of theC5' position to give GDP-
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L-4-keto-gulose. This intermediate is either reduced to give GDP-
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L-gulose orthe C3' position is epimerized to give GDP-
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L-4-keto-galactose, then C4' is reduced to GDP-
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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-
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L-gulose/GDP-
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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
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to the keto function of a carbohydrate identifies key common features.