Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

Abstract

2 β-glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-3 oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition 4 of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with 5 β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in 6 Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in 7 the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and 8 the β-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We 9 identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased 10 hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced 11 substrate accessibility and significant improvement in activity. Analysis of structures and in combination 12 with molecular dynamics simulations show that glucose increases the accessibility of the substrate by 13 enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific 14 activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may 15 offer novel ways of engineering glucose tolerance.