Differences In Gluco And Galacto Substrate-Binding Interactions In A Dual 6p Beta-Glucosidase/6p Beta-Galactosidase Glycoside Hydrolase 1 Enzyme From Bacillus Licheniformis

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-beta-galactosidase and 6-phospho-beta-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-beta-galactosidase/6-phospho-beta-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands p-nitrophenyl-beta-D-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-beta-D-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BlBglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BlBglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BlBglC complex. Additionally, the BlBglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BlBglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.