The structure-function relationship of thermostable beta-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102 / 生物工程学报

Beta-glycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostable monomeric protein and adopts the (beta/alpha)8 barrel fold, is an excellent model system to be investigated for the thermostable mechanism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly (Wang et al, 2003) and comparison of other proteins structure of homofamily, Glu164 and Glu338 may act as proton donor and nucleophile in the hydrolysis reaction respectively; proline located at N1 of alpha-helix and arginine which can form ion link may contribute to the thermostability. 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 had been constructed by site-directed mutagenesis. They are Glu164Gln, Glu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified to SDS-PAGE purity. Changes in the conformations were 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 were determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at N1 positions of alpha-helix, and Arg325 that form salt bridge between alpha-helices 5 and alpha-helices 6, are the critical sites to protein thermostabilization.

Cloning and expression of a thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102 / 生物工程学报

The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E. coli. The gene open reading frame was 1311 bp and it codes for 436 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I. The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%). From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group. The DNASTAR program was used to predict the secondary structure. According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns. 14 of the 35 Pro were located at the second sites of beta-turns. Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability.