| Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28. | |
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MedLine Citation:
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PMID: 23201198 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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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. |
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Authors:
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Preeyanuch Thongpoo; Lauren S McKee; Ana Catarina Araújo; Prachumporn T Kongsaeree; Harry Brumer |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-11-28 |
Journal Detail:
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Title: Biochimica et biophysica acta Volume: - ISSN: 0006-3002 ISO Abbreviation: Biochim. Biophys. Acta Publication Date: 2012 Nov |
Date Detail:
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Created Date: 2012-12-3 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0217513 Medline TA: Biochim Biophys Acta Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2012. Published by Elsevier B.V. |
Affiliation:
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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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