Differences in Gluco and Galacto Substrate-Binding Interactions in a Dual 6Pβ-Glucosidase/6Pβ-Galactosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis

Abstract

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-β-galactosidase and 6-phospho-β-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-β-galactosidase/6-phospho-β-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-β-d-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-β-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.

Dual 6Pβ-Galactosidase/6Pβ-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1

“…These residues are conserved in the 6Pβ-glc 4GPN from S. mutans (Figure 1), and, moreover, the Val residue is described as a residue conserved in all the 6Pβ-glc. 28 The presence in Lp_3525 of typical residues found in 6Pβ-glc and the absence of the tryptophan residue present in 6Pβ-gal (Trp-429 in 4PBG) or in the previously described dual 6Pβ-gal/6Pβ-glc protein (Trp-433 in BlBglC) suggested that Lp_3525 belongs to a different dual 6Pβ-gal/6Pβ-glc protein group (Figure 1). The sequence of Lp_3525 has typical residues conserved in 6Pβ-glc.…”

“…23 This residue is also present in GK3214, a thermostable 6Pβ-glycoside GH1 family from Geobacillus kaustophilus HTA426, 27 which hydrolyzed 6Pβ-gal and 6Pβglc with high activity. Recently, Veldman et al 28 described the differences between gluco and galacto substrate-binding interactions in a dual 6Pβ-gal/6Pβ-glc GH1 enzyme, BlBglC, from Bacillus licheniformis. Moreover, they described the similarities and differences in active-site residue interactions between dual 6Pβ-gal/6Pβ-glc, 6Pβ-gal, and 6Pβ-glc activities, taking BlBglC from B. licheniformis, 4PBG (LacG) from Lactococcus lactis subsp.…”

“…However, BlBglC, GK3214, and Gan1D proteins have been previously described as "dual 6Pβ-gal/6Pβ-glc", 25,27,28 despite presenting a similar sequence identity to 4GPN and 4PBG (35.16 and 40.75% identity, respectively). Moreover, they possess residues and motifs specific for 6Pβ-gal, such as the asparagine and tryptophan residues (Asn-297 and Trp-429 in 4PBG), 25,30 and the lid motif that blocks the entrance to the active site.…”

“…Therefore, so far, the differences in gluco and galacto substrate-binding interactions in dual 6Pβ-gal/6Pβ-glc GH1 glycosidases have only been analyzed in proteins sharing sequence and structural features with 6Pβ-gal (such as BlBglC, GK3214, and Gan1D). 28 However, gathered evidence indicates that there is a different group of GH1 glycosidases possessing dual 6Pβ-gal/6Pβ-glc activity whose the structural basis for their enzyme bifunctionality remains unknown.…”