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PEDS Advance Access published online on May 12, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp015
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Site-directed mutagenesis to probe catalysis by a Thermobifida fusca β-1,3-glucanase (Lam81A)

Colleen E. McGrath1, Thu V. Vuong2 and David B. Wilson2,3

1Department of Biochemistry, Vanderbilt School of Medicine, Nashville, TN 37232, USA 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

3 To whom correspondence should be addressed. E-mail: dbw3{at}cornell.edu

Thermobifida fusca: Lam81A is a single domain family-81 β-1,3-endoglucanase, but no structure is known for this family. Site-directed mutagenesis of 14 conserved residues chosen from sequence alignments was used to identify those with critical roles in catalysis, binding or substrate specificity. Mutant enzymes were assayed for their ability to bind and hydrolyze substrates with various glycosyl linkages. Residues D422, E499 and E503 were candidates for the catalytic acid or catalytic base, and E499 was shown to be the catalytic base by azide rescue. F425 was shown to have a major role in substrate binding possibly mediated by aromatic ring stacking with the sugar substrate. In addition, mutation of D424 to histidine altered the substrate specificity by increasing the rate of cleavage of mixed-linkage β-glucan and carboxymethyl-cellulose, 60- and 16-fold, respectively, over the wild-type enzyme.

Keywords: azide rescue/catalytic base/substrate inhibition/substrate specificity

Received March 13, 2009; revised April 15, 2009; accepted April 16, 2009.


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