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

Darrell Cockburn, Chris Vandenende, Anthony J. Clarke

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining sitedirected mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

Original languageEnglish (US)
Pages (from-to)2042-2050
Number of pages9
JournalBiochemistry
Volume49
Issue number9
DOIs
StatePublished - Mar 9 2010

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Cellulomonas
Cellulase
Aspartic Acid
Substitution reactions
Catalysts
Enzymes
Mutagenesis
Glycoside Hydrolases
Chemical modification
Modulation
Cellulases
Nucleophiles
Acids
Site-Directed Mutagenesis
Hydrogen Peroxide
Databases
Derivatives
Oxidation
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase a from cellulomonas fimi",
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 that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining sitedirected mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.",
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Modulating the pH-activity profile of cellulase by substitution : replacing the general base catalyst aspartate with cysteinesulfinate in cellulase a from cellulomonas fimi. / Cockburn, Darrell; Vandenende, Chris; Clarke, Anthony J.

In: Biochemistry, Vol. 49, No. 9, 09.03.2010, p. 2042-2050.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Modulating the pH-activity profile of cellulase by substitution

T2 - replacing the general base catalyst aspartate with cysteinesulfinate in cellulase a from cellulomonas fimi

AU - Cockburn, Darrell

AU - Vandenende, Chris

AU - Clarke, Anthony J.

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N2 - 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 sitedirected mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

AB - 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 sitedirected mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

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