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Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C-2 epimerization in all nucleotide (CDP, UDP, GDP, ADP, and TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD+/subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a kcat with CDP-d-glucose of ∼1.0 min−1 (pH 7.5 and 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemoenzymatic synthesis for CDP-d-mannose, CDP-6-deoxy-d-glucose, CDP-3-deoxy-d-glucose, and CDP-6-deoxy-d-xylo-hexopyranos-4-ulose. Attempts to obtain CDP-d-paratose and CDP-d-tyvelose were not successful. Using high-resolution carbohydrate analytics and in situ nuclear magnetic resonance (NMR) to monitor the enzymatic conversions (60°C and pH 7.5), we show that the CDP-d-mannose/CDP-d-glucose ratio at equilibrium is 0.67 (±0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C-6 enhances the enzyme activity 5-fold compared to CDP-d-glucose, whereas deoxygenation at C-3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDRs (short-chain dehydrogenases/reductases), for which the current study now provides functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis. IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. The discovery of new epimerases with an expanded scope of sugar nucleotide substrates used is important to promote mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C-2 epimerization in CDP-glucose and other nucleotide-activated forms of d-glucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C-2 epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis.

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GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.