Enhanced GDP-d-mannose 4-epimerization and dynamic insights into NDP-sugar 4-epimerase substrate specificity

UDP-galactose 4-epimerase (Gal4E) catalyze the reversible 4-epimerization of UDP-d-galactose to UDP-d-glucose, a crucial step in galactose metabolism first described by Luis Leloir. Since its initial discovery, a plethora of homologous 4-epimerases has been reported with activities on a variety of other nucleotide sugars, including UDP-D-N-acetyl-galacto/glucosamine, UDP-xylose/l-arabinose, UDP-galacturonic/glucuronic acid, dTDP-l-rhamnose/6-deoxy-l-talose and/or GDP-hexoses. These NDP-sugar 4-epimerases (NS-4Es) play critical roles in diverse carbohydrate metabolic pathways across all domains of life and exhibit conserved structures and mechanism. Hence, various models have sought to predict specificity by focusing on steric constraints and conserved motifs, with successful specificity switches in Gal4Es based on and supporting the gatekeeper model. However, mutational analysis in other representatives also challenge these models, suggesting that additional factors like molecular interactions and conformational dynamics may significantly influence substrate specificity. This study combines sequence similarity networks, site saturation mutagenesis and molecular dynamics to investigate substrate specificity in NS-4Es. We propose an expanded classification, increasing specificity groups from three to seven to incorporate epimerases active on GDP-sugars (group 4), UDP-d-xylose (group 5), UDP-d-galacturonic acid (group 6), and dTDP-l-rhamnose (group 7). Mutagenesis of the gatekeeper in GDP-hexose 4-epimerase PhGal4E_1 revealed enhanced activity on GDP-D-Man, which was found to be driven both by gatekeeper size and polarity as well as by altered active site interactions. Molecular dynamics confirmed a dynamic network and clamping down mechanism within the active site as key determinants of specificity across NS-4Es, offering new insights into their functional diversity.