Structure and Function of GDP-Mannose-3‘,5‘-Epimerase:  An Enzyme which Performs Three Chemical Reactions at the Same Active Site

Abstract

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-Lgalactose. 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.

The Crystal Structure of Progesterone 5β-Reductase from Digitalis lanata Defines a Novel Class of Short Chain Dehydrogenases/Reductases

“…1B and Table 2). As almost always observed in dinucleotide-binding proteins, a conserved water molecule bridges the interactions between the glycine-rich sequence and the diphosphate group (41,42). This water molecule is numbered water molecule 1 in 5␤-POR in complex with NADP ϩ because it displays the highest electron density of all water molecules.…”

“…This proposal is corroborated by the observation that in sugar epimerases where the reaction is considered to proceed via a 1-2 hydrogen subtraction reaction by means of a temporary oxidation of a hydroxyl group to a keto-intermediate, the standard SDR orientation of the residues lysine and tyrosine is adopted, although all other substrate protein interactions differ considerably (42). On the other hand in enoyl-reductases (48), which catalyze a 1-4 hydrogen addition reaction similarly to 5␤-POR, the active site tyrosine side chain is also repositioned with respect to the lysine residue and the nicotinamide ring in the active site because the conserved YXXXK motif is replaced by a YXXMXXXK motif.…”

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

“…GDP-α-L-Gal (GDP-Gal) was enzymatically synthesized according to previously outlined methods (39) and HPLCpurified using a linear ammonium formate gradient (40). UDP-β-L-Rha was enzymatically synthesized by a two-step reaction using UDP-α-D-Glc (UDP-Glc) as a substrate.…”

Proteins involved in biotic and abiotic stress responses as the most significant biomarkers in the ripening of Pinot Noir skins

“…GDP-mannose-3′,5′-epimerase (ManEp) catalyzes the reversible conversion of GDP-D-mannose to GDP-Lgalactose and to GDP-L-gulose and it was suggested that this reaction is a key regulatory step of ascorbic acid (AsA) biosynthesis (Wolucka et al 2001;Major et al 2005). It has been recently indicated that in a comparison among kiwifruit genotypes that differ for the levels of AsA, the concentration of the vitamin is correlated to the high amount of GDP-mannose-3′,5′-epimerase and GDP-Lgalactose guanyltransferase also because, overexpressing the genes of the two enzymes in transgenic kiwifruit plants, a significant increase in AsA levels occurred (Bulley et al 2009).…”