期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1814, 期 12, 页码 1616-1623出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2011.08.012
关键词
beta-glucosidase; Glycoside hydrolase; Substrate specificity; Contact pathways
资金
- FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) [08/55914-9, 08/57619-4]
- CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)
- Instituto Nacional de Ciencias e Tecnologia para o Bioetanol [08/5798-1, 574002/2008-1]
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [08/55914-9] Funding Source: FAPESP
A library of random mutants of the beta-glycosidase Sf beta gly was screened for mutations that affect its specificity for the substrate glycone (beta-D-fucoside versus beta-D-glucoside). Among mutations selected (T35A, R189G, Y345C, P348L, S358F, S378G, N400D, S424F, F460L, and R474H), eight occurred in the C-terminal half of Sf beta gly and only two were at the active site (R189G and N400D). Tryptophan fluorescence spectra and thermal inactivation showed that the selected mutants and wild-type Sf beta gly are similarly folded. Enzyme kinetics confirmed that these mutations resulted in broadening or narrowing of the preference for the substrate glycone. Structural modeling and interaction maps revealed contact pathways that connect the sites of the selected mutations through up to three interactions to the active site residues E399, W444, and E187, which are involved in substrate binding and catalysis. Interestingly, independently selected mutations (Y345C, P3481, and R189G; S424F and N400D) were placed on the same contact pathway. Moreover, (k(cat)/K-m fucoside)/(k(cat)/K-m glucoside) ratios showed that mutations at intermediate residues of the same contact pathway often had similar effects on substrate specificity. Finally mutations in the same contact pathway caused similar structural disturbance as evidenced by acrylamide quenching of the Sf beta gly fluorescence. Based on these data, it is proposed that the effects of the selected mutations were propagated into the active site through groups of interacting residues (contact pathways) changing the Sf beta gly substrate specificity. (C) 2011 Elsevier B.V. All rights reserved.
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