The Conversion of UDP-Glc to UDP-Man: In Silico and Biochemical Exploration To Improve the Catalytic Efficiency of CDP-Tyvelose C2-Epimerases

A promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus (TaTyvE) belonging to the nucleotide sugar active short-chain dehydrogenase/reductase superfamily (NS-SDRs) was recently discovered. TaTyvE performs the slow conversion of NDP-glucose (NDP-Glc) to NDP-mannose (NDP-Man). Here, we present the sequence fingerprints that are indicative of the conversion of UDP-Glc to UDP-Man in TyvE-like enzymes based on the heptagonal box motifs. Our data-mining approach led to the identification of 11 additional TyvE-like enzymes for the conversion of UDP-Glc to UDP-Man. We characterized the top two wild-type candidates, which show a 15- and 20-fold improved catalytic efficiency, respectively, on UDP-Glc compared to TaTyvE. In addition, we present a quadruple variant of one of the identified enzymes with a 70-fold improved catalytic efficiency on UDP-Glc compared to TaTyvE. These findings could help the design of new nucleotide production pathways starting from a cheap sugar substrate like glucose or sucrose. Promiscuous CDP-tyvelose 2-epimerase (TyvE) converts NDP-glucose to NDP-mannose. We present the sequence fingerprints that are indicative of this conversion in TyvE-like enzymes. Eleven TyvE-like enzymes were identified, and the top two wild-type candidates and a quadruple mutant were characterized. The improved catalytic efficiency of these enzymes might help the design of new nucleotide production pathways starting from a cheap sugar substrates like sucrose.image

Automated summary

We discovered a promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus that converts NDP-glucose to NDP-mannose. By identifying sequence fingerprints, we found 11 additional TyvE-like enzymes that can convert UDP-glucose to UDP-mannose. We characterized the top two wild-type candidates and a quadruple mutant, which showed improved catalytic efficiency on UDP-glucose. These findings could be valuable for designing nucleotide production pathways using inexpensive sugar substrates like sucrose.