Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: the CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion

Christian Rapp, Stevie van Overtveldt, Koen Beerens, Tom Desmet, Bernd Nidetzky*, Hansjörg Weber

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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 k cat with CDP-D-glucose of ∼1.0 min −1 (pH 7.5 and60°C). To characterizetheepimerasekinetically andprobeits substratespecificity, 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.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalApplied and Environmental Microbiology
Issue number4
Publication statusPublished - Feb 2021


  • carbohydrate synthesis
  • catalytic mechanism
  • epimerase
  • multistep enzyme catalysis
  • short-chain dehydrogenase/reductase (SDR)
  • sugar nucleotide
  • CDP-glucose
  • specificity

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

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