A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases

beta-Glucosidases are enzymes that hydrolyze beta-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, and thus have significant application potential in industries. However, most beta-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some beta-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 beta-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 beta-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 beta-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 beta-glycosidases and related enzymes that modulate their activity via feedback control mechanism.