Substrate (aglycone) specificity of human cytosolic β-glucosidase

Human cytosolic β-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays β-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (β/α)₈-barrel fold characteristic of family 1 β-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val¹⁶⁸→Tyr, Phe²²⁵→Ser, Tyr³⁰⁸→Ala and Tyr³⁰⁸→Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val¹⁶⁸→Tyr mutation did not affect K_m on p-nitrophenyl (pNP)-glycosides, but increased K_m 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe²²⁵→Ser and Tyr³⁰⁸→Ala mutations, and, to a lesser degree, the Tyr³⁰⁸→Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.