Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.
Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Darrell W
DW
Cockburn
Chris
C
Vandenende
Anthony J
AJ
Clarke
D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed
D001426:Bacterial Proteins; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; D003545:Cysteine; C013461:cysteine sulfinic acid