| Summary: | GDP-fucose synthase (GFS), also known as GDP-4-keto-6-deoxy-mannose 3, 5- epimerase / 4-reductase (GMER), is the key enzyme in the GDP-L-fucose biosynthetic pathway. The pathway begins with a dehydratase that converts GDP-D-mannose into 6-deoxy-4-keto- GDP-D-mannose. Then GFS performs two consecutive epirnerizations, and a final NADPH dependent reduction to generate GDP-L-fucose. The detailed mechanism of GFS has remained a mystery due to limited mechanistic studies as well as the lack of an x-ray crystal structure with any bound GDP-sugar. This thesis investigates the mechanism of the reaction as well as the roles of the active site residues in GFS. The results support a sequential ordered epimerization mechanism where the stereocenter at C-3”is inverted first, followed by the inversion of
stereochemistry at C-5”. This identifies GDP-6-deoxy-D-altrose as a reaction intermediate for the first time. The findings of this thesis also leads to the assignment of the catalytic residue Cys 109,
as the base which deprotonates both epimerization sites, and His179 as the acid which reprotonates the corresponding enol intermediates. GDP-mannose 3, 5-epimerase (GME), is the key enzyme that catalyzes the first committed step of vitamin C biosynthesis in plants. It oxidizes the C-4” position of GDP mannose, then sequentially inverts the stereochemistry at C-5” and C-3”, and finally reduces the C-4” carbonyl to produce GDP-L-galactose. The proposed sequential order of epimerization is based on the observation that GDP-L-gulose (the C-5” epimeric product) is produced whereas GDP-D-altrose (the C-3” epimeric product) is not. Structural studies on GME suggest that Cys145 acts as the catalytic base and Lys 217 acts as the catalytic acid in both epimerization steps. However, very limited mechanistic studies on GME have been performed. This thesis provides mechanistic evidence that supports the proposed sequential epimerization order as well as the assignment of roles for the catalytic residues. It also suggests a model whereby protonated Cys145 is unable to exchange its proton with solvent during the lifetime of the enol(late) intermediate.
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