Exploration of archaeal nucleotide sugar epimerases unveils a new and highly promiscuous GDP-Gal4E subgroup

Abstract

• Promiscuity of phGal4E_1 on different NDP-sugars was explored. • First demonstration of C4 epimerization activity on GDP-L-galactose and GDP-L-fucose. • PhGal4E_1, afGal4E and pcGal4E could form a new subgroup within CEP1 C4-epimerases. • The heptagonal box model allows the identification of new specificities in CEP1 family enzymes. UDP-galactose 4-epimerase (Gal4E, EC 5.1.3.2) catalyses the interconversion of UDP-galactose and UDP-glucose by inverting the configuration of the 4’-hydroxyl group of the sugar moiety. This enzyme is one of the mechanistically best-characterized members of the Short-chain Dehydrogenase/Reductase (SDR) superfamily. Although Gal4E was widely studied throughout all domains of life, ranging from eukaryotes to archaea, its biochemical characterization was often limited to UDP-hexoses, neglecting the possibility that Gal4E might be promiscuous towards other NDP-sugars and derivatives thereof. In this study, we identified a novel Gal4E subgroup displaying an unprecedented specificity on guanosine diphosphate (GDP) sugars. As a proof of concept, a detailed biochemical investigation was performed on Gal4E from Pyrococcus horikoshii (phGal4E_1), which revealed that it has, in fact, a clear preference for GDP-sugars. In addition, we confirmed that it accepts a variety of other sugar moieties, including L-sugars like L-galactose and L-fucose.