Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi

Biochemistry
Darrell W CockburnA J Clarke

Abstract

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 t...Continue Reading

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Citations

Jan 29, 2011·Protein Engineering, Design & Selection : PEDS·Darrell W Cockburn, Anthony J Clarke
Feb 22, 2012·Chembiochem : a European Journal of Chemical Biology·John M Pfeffer, Anthony J Clarke
Jun 17, 2010·Biotechnology and Bioengineering·Thu V Vuong, David B Wilson
May 4, 2016·Cellular and Molecular Life Sciences : CMLS·Masayuki OkuyamaAtsuo Kimura
Oct 22, 2019·Biotechnology and Bioengineering·Nanna RøjelPeter Westh
Oct 6, 2017·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Hirak SaxenaWarren Wakarchuk

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