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Improving the oxidative stability of a high redox potential fungal peroxidase by rational design.

Sáez-Jiménez V, Acebes S, Guallar V, Martínez AT, Ruiz-Dueñas FJ - PLoS ONE (2015)

Bottom Line: Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate.Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold.This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.

No MeSH data available.


Turnover studies in the presence of excess of hydrogen peroxide.Kinetics of native VP (A) and its T45A/I103T/M152F/M262F/M265L variant (B) in the presence of a stoichiometric excess of 5000 equivalents of H2O2. Spectra were recorded at 25°C during 20 s. The dashed arrows indicate the increase and decrease of absorbance during formation of CII (with a maximum at 417 nm) and CIII (with maxima at 419 nm, 546 nm and 581 nm, and a minimum at 651 nm), and the decrease of absorbance during heme bleaching (at 419 nm). Traces correspond respectively to 0.01 s (CI spectrum both in A and B, showed as a dashed-point line); 0.1, 0.25, 0.5, 1, 2 and 5 s (continuous lines); and 10 and 20 s (continuous lines in B, and discontinuous lines in A due to heme bleaching). Details of the 500 nm-700 nm region are shown in x8 scale.
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pone.0124750.g006: Turnover studies in the presence of excess of hydrogen peroxide.Kinetics of native VP (A) and its T45A/I103T/M152F/M262F/M265L variant (B) in the presence of a stoichiometric excess of 5000 equivalents of H2O2. Spectra were recorded at 25°C during 20 s. The dashed arrows indicate the increase and decrease of absorbance during formation of CII (with a maximum at 417 nm) and CIII (with maxima at 419 nm, 546 nm and 581 nm, and a minimum at 651 nm), and the decrease of absorbance during heme bleaching (at 419 nm). Traces correspond respectively to 0.01 s (CI spectrum both in A and B, showed as a dashed-point line); 0.1, 0.25, 0.5, 1, 2 and 5 s (continuous lines); and 10 and 20 s (continuous lines in B, and discontinuous lines in A due to heme bleaching). Details of the 500 nm-700 nm region are shown in x8 scale.

Mentions: The spectral changes of the native VP and of three variants improving oxidative stability (M152F/M262F/M265L, T45A/I103T and T45A/I103T/M152F/M262F/M265L) were analyzed after enzyme activation in the presence of 5000 equivalents of H2O2 (high enough to see the transitions between the three states of the catalytic cycle in a short period of time). The first spectrum of the native enzyme, obtained at 10 ms, exhibited typical maxima of CI including the Soret band at 403 nm and the charge transfer band CT1 at 651nm (Fig 6A). Subsequently, CII was formed as observed by the displacement of the Soret band to 417 nm, the reorganization of the 500–600 nm spectral region and the disappearance of the maximum at 651 nm. Then CIII was generated exhibiting a characteristic spectrum with maxima at 419, 546 and 581 nm. Comparable spectral changes, although with different conversion rates, were obtained for the VP variants, as illustrated for the T45A/I103T/M152F/M262F/M265L variant in Fig 6B. When the reaction was extended in time (up to 20 min) strong differences in CIII decay and enzyme bleaching were observed (Fig 7), as discussed below together with the differences in CIII formation.


Improving the oxidative stability of a high redox potential fungal peroxidase by rational design.

Sáez-Jiménez V, Acebes S, Guallar V, Martínez AT, Ruiz-Dueñas FJ - PLoS ONE (2015)

Turnover studies in the presence of excess of hydrogen peroxide.Kinetics of native VP (A) and its T45A/I103T/M152F/M262F/M265L variant (B) in the presence of a stoichiometric excess of 5000 equivalents of H2O2. Spectra were recorded at 25°C during 20 s. The dashed arrows indicate the increase and decrease of absorbance during formation of CII (with a maximum at 417 nm) and CIII (with maxima at 419 nm, 546 nm and 581 nm, and a minimum at 651 nm), and the decrease of absorbance during heme bleaching (at 419 nm). Traces correspond respectively to 0.01 s (CI spectrum both in A and B, showed as a dashed-point line); 0.1, 0.25, 0.5, 1, 2 and 5 s (continuous lines); and 10 and 20 s (continuous lines in B, and discontinuous lines in A due to heme bleaching). Details of the 500 nm-700 nm region are shown in x8 scale.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414599&req=5

pone.0124750.g006: Turnover studies in the presence of excess of hydrogen peroxide.Kinetics of native VP (A) and its T45A/I103T/M152F/M262F/M265L variant (B) in the presence of a stoichiometric excess of 5000 equivalents of H2O2. Spectra were recorded at 25°C during 20 s. The dashed arrows indicate the increase and decrease of absorbance during formation of CII (with a maximum at 417 nm) and CIII (with maxima at 419 nm, 546 nm and 581 nm, and a minimum at 651 nm), and the decrease of absorbance during heme bleaching (at 419 nm). Traces correspond respectively to 0.01 s (CI spectrum both in A and B, showed as a dashed-point line); 0.1, 0.25, 0.5, 1, 2 and 5 s (continuous lines); and 10 and 20 s (continuous lines in B, and discontinuous lines in A due to heme bleaching). Details of the 500 nm-700 nm region are shown in x8 scale.
Mentions: The spectral changes of the native VP and of three variants improving oxidative stability (M152F/M262F/M265L, T45A/I103T and T45A/I103T/M152F/M262F/M265L) were analyzed after enzyme activation in the presence of 5000 equivalents of H2O2 (high enough to see the transitions between the three states of the catalytic cycle in a short period of time). The first spectrum of the native enzyme, obtained at 10 ms, exhibited typical maxima of CI including the Soret band at 403 nm and the charge transfer band CT1 at 651nm (Fig 6A). Subsequently, CII was formed as observed by the displacement of the Soret band to 417 nm, the reorganization of the 500–600 nm spectral region and the disappearance of the maximum at 651 nm. Then CIII was generated exhibiting a characteristic spectrum with maxima at 419, 546 and 581 nm. Comparable spectral changes, although with different conversion rates, were obtained for the VP variants, as illustrated for the T45A/I103T/M152F/M262F/M265L variant in Fig 6B. When the reaction was extended in time (up to 20 min) strong differences in CIII decay and enzyme bleaching were observed (Fig 7), as discussed below together with the differences in CIII formation.

Bottom Line: Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate.Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold.This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.

No MeSH data available.