Sets of Covariant Residues Modulate the Activity and Thermal Stability of GH1 β-Glucosidases

The statistical coupling analysis of 768 beta-glucosidases from the GH1 family revealed 23 positions in which the amino acid frequencies are coupled. The roles of these covariant positions in terms of the properties of b-glucosidases were investigated by alanine-screening mutagenesis using the fall armyworm Spodoptera frugiperda beta-glycosidase (Sf beta gly) as a model. The effects of the mutations on the Sf beta gly kinetic parameters (k(cat)/K-m) for the hydrolysis of three different p-nitrophenyl beta-glycosides and structural comparisons of several b-glucosidases showed that eleven covariant positions (54, 98, 143, 188, 195, 196, 203, 398, 451, 452 and 460 in Sfbgly numbering) form a layer surrounding the active site of the beta-glucosidases, which modulates their catalytic activity and substrate specificity via direct contact with the active site residues. Moreover, the influence of the mutations on the transition temperature (T-m) of Sfbgly indicated that nine of the coupled positions (49, 62, 143, 188, 223, 278, 309, 452 and 460 in Sfbgly numbering) are related to thermal stability. In addition to being preferentially occupied by prolines, structural comparisons indicated that these positions are concentrated at loop segments of the b-glucosidases. Therefore, due to these common biochemical and structural properties, these nine covariant positions, even without physical contacts among them, seem to jointly modulate the thermal stability of beta-glucosidases.