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Structure-function relationships in a f3-1,4-glycanase (Cex) from Cellulomonas fimi: identification of catalytic residues

University of British Columbia

The objective of this study was to identify and examine the roles of catalytic residues in the active site of a ß-1,4-glycanase (Cex) from the bacterium Cellulomonas fimi. The use of the Gram positive bacterium, Streptomyces lividans, as a host for the expression of the cex gene was explored. The cex gene was successfully expressed in S. lividans from the aph promoter of p1J680-cex. The polypeptide was efficiently exported to the culture medium. Yields of recombinant polypeptide were about 6 mg/L following 40 hours of growth. Cex produced by S. lividans had a molecular mass greater than that produced by E. coli due to glycosylation. Similar to the native enzyme from C. fimi, the glycosylation protected the enzyme from a C. fimi protease, particularly when the enzyme was bound to cellulose. Cex hydrolyses ß-1,4-glycosidic bonds in cellulose with retention of anomeric configuration, releasing 13-cellobiose. On the basis of amino acid sequence alignments, Glu 233 was proposed as the catalytic nucleophile in Cex. The kinetic parameters determined for Glu 233 mutants generated by site-directed mutagenesis were consistent with this interpretation. Similarly, Glu 127 was proposed as the acid/base catalyst in Cex. The kinetic parameters were determined for Glu 127 mutants generated by site-directed mutagenesis, using a range of soluble cellobiosides and glucosides with differing requirements for acid catalysis. The results were consistent with a role of Glu 127 as acid/base catalyst. In the presence of azide, a new product, ß-cellobiosyl-azide was formed with uncharged Glu 127 mutants and catalytic activity was restored to wild-type levels. The results suggest azide occupied a vacant anionic site consistent with the removal of the acid/base catalyst. The approach used in this study could be applied to the identification of the acid/base catalyst in other ß-l,4-glycanases, particularly in the absence of threedimensional structure information.

Thesis/Dissertation

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2009-04-14

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http://hdl.handle.net/2429/7041

Genetics

University of British Columbia

UBCV

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