Abstract
Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (Vitis vinifera) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a V. vinifera aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer–specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in Arabidopsis thaliana. Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer–specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer–specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants.
Highlights
The enzyme assay in our study showed that Vv2KGR could utilize both NADH and NADPH as cofactor but displayed a clear preference for NADPH (Fig. 2)
We have identified an aldo-keto reductase Vv2KGR with 2-keto-L-gulonic acid reductase activity from grapevine
The transcription profile of Vv2KGR was consistent with TA accumulation in developing grape berries, supporting its potential role in TA biosynthesis from vitamin C in grapevine
Summary
Best of our knowledge, this is the first report of a D-isomer–specific 2-hydroxyacid dehydrogenase that reduces 2-keto-L-gulonic acid to L-idonic acid in plants. L-threo-Tetruronate is oxidized to TA while the two-carbon fragment, possibly glycoaldehyde, is recycled into triose and hexose phosphate metabolism [23, 24] In this pathway, the conversion of IA to 5-keto-D-gluconic acid has been proposed as the rate-limiting step [5]. Enzymes with the ability to catalyze the same chemical reaction have been reported in a number of microorganisms, including Erwinia herbicola [26], Brevibacterium ketosoreductum [27], Escherichia coli [28], and various acetic acid bacteria [29] In these microbes, 2KGRs are able to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, 2-keto-D-gluconate to D-gluconate, and 2KLG to IA, playing a role in the ketogluconate metabolic pathway (26 –28). Our study is the first to report that a 2KDH can convert 2KLG to IA in plants
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