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Functional and mutational analysis of the cellulose binding domain CBDN1 from Cellulomonas fimi [Beta]-glucanase C (CenC)

University of British Columbia

Endoglucanase C (CenC), a ß,1-4 glucanase from the soil bacterium Cellulomonas fimi binds to cellulose via the cellulose binding domains N l (CBDN1), and N2 (CBDN2). In this thesis, the contribution of 10 amino acids within the binding cleft of CBDN1 was evaluated by alanine mutagenesis. Each mutant was analyzed for binding to an insoluble allomorph of cellulose (phosphoric acid swollen Avicel), and a soluble glucopyranoside polymer (barley ß-glucan). All ten mutations affected the binding affinity of CBDN1 to some extent; Y19 and Y85 were identified as the two residues most essential for tight binding to insoluble cellulose and barley ß-glucan. The structural integrity and intermolecular contacts of Y19A and Y85A were assessed using two-dimensional NMR spectrospcopy and UV resonance Raman spectroscopy (UVRRS). Both techniques indicated that Y85A is similar to wild-type CBDN1 in structure, but that Y19A exhibits some structural perturbations in the binding cleft. In addition, UVRRS data indicated that Y85 forms a direct hydrogen bond to cellulose upon binding, but Y19 does not. Taken together, these data imply that CBDN1 binding is mediated by two tyrosine residues, one of which interacts directly with cellulose, the other important for structural integrity of the binding cleft.

Thesis/Dissertation

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2009-05-19

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http://hdl.handle.net/2429/7927

Microbiology and Immunology

University of British Columbia

UBCV

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