Differences in Gluco and Galacto Substrate-Binding
Interactions in a Dual 6Pβ-Glucosidase/6Pβ-Galactosidase
Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis
posted on 2021-08-23, 15:33authored byWayde Veldman, Marcelo Vizona Liberato, Valquiria P. Souza, Vitor M. Almeida, Sandro R. Marana, Özlem Tastan Bishop, Igor Polikarpov
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.