Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) beta-glucosidase from Aspergillus niger ASKU28

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

beta-Glucosidase

Acid/base catalyst

Aspergillus niger

Glycoside hydrolase family 3

Azide rescue

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)