Structure and function of GDP-mannose-3′ ,5′ -epimerase; an enzyme which performs three chemical reactions at the same active site

By L. Major, B. A. Wolucka, J. Naismith. 2005

89 citations recorded by the source corpus.

GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana catalyses 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 Å 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. Based on 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.

Subfields: Materials Chemistry, Molecular Biology