The Structure-function Relationship Of Thermostable Beta-Glycosidase From The Thermophilic Eubacterium Thermus Nonproteolyticus HG102

β-Glycosidase (Tn-gly) from the thermophilic eubacterium Thermus nonproteolyticus HG102 which is a thermostable monomeric protein and adopts the (β/a)8 barrel fold, is an excellent model system to be investigated the thermostable mechanism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tn-gly (Wang, et al, 2003) and comparison of other proteins structure of homofamily, Glul64 and Glu338 may act as proton donor and nucleophile in the hydrolysis reaction respectively; proline located at Nl of a-helix and arginine which can form ion link may contribute to the thermostability; Gln 18, Hisl20, Glu392, Asn282, Tyr284 and Trp385 are hypothesized to determine substrate specificity. We aim to further identify the critical sites and the amino acid residue(s) responsible for the activity , the thermal stability and the substrate specificity. Mutations have been constructed by site-directed mutagenesis. They are Glul64Gln, Glu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified. Changes in the conformations was examined by means of CD. The Glu338Ala mutant showed no detectable hydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. The Glu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-type was determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline which is located at Nl positions of a-helix, and Arg325 which form salt bridge between a helices 5 and a helices 6, are the critical sites to protein thermostabilization.