title: [The structure-function relationship of thermostable beta-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102].
abstract: 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.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Science, Beijing 100080, China.
authors.forename: Xue-Peng
authors.initials: XP
authors.lastname: Yang
authors.forename: Shou-Jun
authors.initials: SJ
authors.lastname: Yang
authors.forename: Bei-Zhong
authors.initials: BZ
authors.lastname: Han
authors.forename: Cheng
authors.initials: C
authors.lastname: Jin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013329:Structure-Activity Relationship; D013824:Thermus / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001617:beta-Glucosidase; C441633:beta-glycosidase, Thermus nonproteolyticus