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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Abstract
GDP-mannose-3‘,5‘-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3‘ and 5‘ positions of GDP-α-d-mannose to yield GDP-β-l-galactose. Production of the C5‘ epimer of GDP-α-d-mannose, GDP-β-l-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-α-d-mannose, GDP-β-l-galactose, and a mixture of GDP-β-l-gulose with GDP-β-l-4-keto-gulose to resolutions varying from 2.0 to 1.4 Å. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-β-l-galactose and GDP-β-l-gulose. The reaction proceeds by C4‘ oxidation of GDP-α-d-mannose followed by epimerization of the C5‘ position to give GDP-β-l-4-keto-gulose. This intermediate is either reduced to give GDP-β-l-gulose or the C3‘ position is epimerized to give GDP-β-l-4-keto-galactose, then C4‘ is reduced to GDP-β-l-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-β-l-gulose/GDP-β-l-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein α to the keto function of a carbohydrate identifies key common features.