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Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) beta-glucosidase from Aspergillus niger ASKU28
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.ORCID iD: 0000-0002-3372-8773
KTH, School of Biotechnology (BIO), Glycoscience.
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2013 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1830, no 3, 2739-2749 p.Article in journal (Refereed) Published
Abstract [en]

Background: The commercially important glycoside hydrolase family 3 (GH3) beta-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 beta-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 beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on add 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 beta-glucosides and yielded beta-glucosyl azide as a product. Conclusions and general significance: These results strongly support the assignment of E490 as the acid/base catalyst in a beta-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.

Place, publisher, year, edition, pages
2013. Vol. 1830, no 3, 2739-2749 p.
Keyword [en]
beta-Glucosidase, Acid/base catalyst, Aspergillus niger, Glycoside hydrolase family 3, Azide rescue
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-119724DOI: 10.1016/j.bbagen.2012.11.014ISI: 000315307900026ScopusID: 2-s2.0-84873577162OAI: diva2:612778
FormasKnut and Alice Wallenberg Foundation

QC 20130325

Available from: 2013-03-25 Created: 2013-03-21 Last updated: 2013-03-25Bibliographically approved

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Thongpoo, PreeyanuchMcKee, Lauren S.Araújo, Ana CatarinaBrumer, Harry
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