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Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28.
MedLine Citation:
PMID:  23201198     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
BACKGROUND: The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined. METHODS: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28. RESULTS: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K(m) values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-D-glucoside and 2,4-dinitrophenyl β-D-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product. Conclusions and General Significance These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.
Authors:
Preeyanuch Thongpoo; Lauren S McKee; Ana Catarina Araújo; Prachumporn T Kongsaeree; Harry Brumer
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-28
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  -     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-12-3     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier B.V.
Affiliation:
Interdisciplinary Graduate Program in Genetic Engineering, Faculty of Graduate School, Kasetsart University, Bangkok, 10900, Thailand; Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden. Electronic address: g5089003@ku.ac.th.
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