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

Chembiochem

Centre for Synthetic Biology (CSB), Unit for Biocatalysis and Enzyme Engineering, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Gent, Belgium.

Published: December 2023

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.

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http://dx.doi.org/10.1002/cbic.202300549DOI Listing

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