CDP-paratose 2-epimerase: substrate specificity beyond expectation

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CDP-paratose 2-epimerase: substrate specificity beyond expectation

Christian Rapp 1 , Stevie Van Overtveldt 3 , Koen Beerens 3 , Tom Desmet 3 , Bernd Nidetzky 1,2

1 Institute Of Biotechnology And Biochemical Engineering, Graz University Of Technology, Graz, Austria, 2 Austrian Centre of Industrial Biotechnology (acib), Graz, Austria, 3 Centre for Synthetic Biology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium

Carbohydrate 2-epimerases have evolved to invert the stereochemistry at C-2 on their respective sugar substrates following various catalytic mechanisms. Those mechanisms involve deprotonation/reprotonation, nucleotide elimination/readdition or formation of a transient keto-intermediate, the latter being proposed for CDP-paratose 2-epimerase. An uncharacterized, dimeric homologue of the aforementioned CDP-paratose 2-epimerase (TyvE) originating from Thermodesulfatator atlanticus displaying an expanded substrate scope is sculpting the frame of this research: CDP-glucose was found to function as alternate, readily accessible substrate. The epimerization to CDP-mannose was confirmed by in situ NMR studies as well as the chemo-enzymatic synthesis of CDP-mannose applied in the back-reaction. Catalytic efficiencies (kcat,f/Km,f ≃47.42 [1/(Ms)], kcat,b/Km,b ≃68.23 [1/(Ms)]) corresponding to a Haldane relationship of Keq,H ≃0.70 are in accordance to an equilibrium constant of Keq ≃0.67 indicating a thermodynamically disfavored forward-reaction. TyvE showing a temperature optimum of 70°C further enabled the observation of kinetic isotope effects at C-2 over a temperature range of 20°C to 80°C resulting in an average KIE of ≃2.5.

This research is part of the EpiSwitch project, jointly funded by the Fund for Scientific Research Flanders (FWO-Vlaanderen, grant n° G0F3417N) and the Austrian Science Fund (FWF; project n° I 3247).

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