CDP-paratose 2-epimerase: substrate specificity beyond expectation

S2.4 Sugar biosynthesis
Location (hall): 
Start/end time: 
Monday, July 1, 2019 - 14:45 to 15:00

Christian Rapp1, Stevie Van Overtveldt3, Koen Beerens3, Tom Desmet3, Bernd Nidetzky1,2

1Institute Of Biotechnology And Biochemical Engineering, Graz University Of Technology, Graz, Austria, 2Austrian Centre of Industrial Biotechnology (acib), Graz, Austria, 3Centre 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).