Structural Superimposition

Abstact

UDP-glucose 4-epimerase (GalE) is a key enzyme in the Leloir pathway that catalyzes the reversible conversion of UDP-glucose to UDP-galactose and plays a central role in galactose metabolism. To investigate the structural basis of this reaction in cyanobacteria, the protein encoded by all4713 from Nostoc sp. PCC 7120 was identified and characterized as a GalE enzyme. The protein was crystallized in complex with UDP-glucose and NAD(+), and its structure revealed a typical epimerase fold consisting of nine alpha-helices and a seven-stranded beta-sheet that together form the catalytic pocket accommodating both ligands. To probe the catalytic mechanism, seven active-site variants (A84K, T124A, T124S, A126T, A150K, H196 N, and L296 M) were generated by site-directed mutagenesis. Enzymatic assays indicated that the L296 M variant exhibited higher activity than the wild-type enzyme under the tested conditions, suggesting that this residue may influence substrate interactions within the catalytic site. Based on these findings, an enzymatic route for galactose generation was outlined in which sucrose synthase (SuSy) produces UDP-glucose from sucrose and UDP, followed by GalE-mediated conversion to UDP-galactose and subsequent hydrolysis to yield galactose. The resulting galactose can be further converted to tagatose by L-arabinose isomerase. This work provides structural and functional insight into the GalE enzyme from Nostoc and supports its potential application in enzymatic sugar conversion processes.