Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity

Brugger, Dagmar; Sützl, Leander; Zahma, Kawah; Haltrich, Dietmar; Peterbauer, Clemens K.; Stoica, Leonard

doi:10.1039/c6cp06009a

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…structural comparison with glucose oxidase and cholesterol oxidase, for which the corresponding position was altered to reduce oxygen activity 49 T747A, T747H found as important during previous studies aiming at enhancing oxygen activity of CDH N748X, N748C N748 coordinates FAD and modulates oxygen activity; 70 pyranose 2-oxidase equivalent N593C turned oxidase into dehydrogenase 71 structural stability M75X M75 complexes the heme cofactor 7 M309X mass spectrometry data indicated a high degree of oxidation during substrate turnover within the first hours (Figure S2) M409X close to FAD M513X oxidation was not observed during substrate turnover by mass spectrometry (Figure S2), hence unclear potential M581X in molecular dynamics simulations, oxygen was attracted to a hydrophobic patch of CDH; together with F326 and L324, M581 forms a hydrophobic pocket M690X, M690L, M690Y…”

Section: Y619xmentioning

confidence: 99%

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Geiss

Reichhart

Pejker

et al. 2021

ACS Sustainable Chem. Eng.

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Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general.

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Section: Y619xmentioning

confidence: 99%

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Geiss

Reichhart

Pejker

et al. 2021

ACS Sustainable Chem. Eng.

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“…The second category focuses on a cavity in the vicinity of the isoalloxazine ring [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Considering the reaction between oxygen and flavin, the region is likely crucial for the oxidative half-reaction.…”

Section: Engineering Of the Oxidative Half-reaction With Oxygenmentioning

confidence: 99%

Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases

Hiraka

Tsugawa

Sode

2020

IJMS

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In this review, recent progress in the engineering of the oxidative half-reaction of flavin-dependent oxidases and dehydrogenases is discussed, considering their current and future applications in bioelectrochemical studies, such as for the development of biosensors and biofuel cells. There have been two approaches in the studies of oxidative half-reaction: engineering of the oxidative half-reaction with oxygen, and engineering of the preference for artificial electron acceptors. The challenges for engineering oxidative half-reactions with oxygen are further categorized into the following approaches: (1) mutation to the putative residues that compose the cavity where oxygen may be located, (2) investigation of the vicinities where the reaction with oxygen may take place, and (3) investigation of possible oxygen access routes to the isoalloxazine ring. Among these approaches, introducing a mutation at the oxygen access route to the isoalloxazine ring represents the most versatile and effective strategy. Studies to engineer the preference of artificial electron acceptors are categorized into three different approaches: (1) engineering of the charge at the residues around the substrate entrance, (2) engineering of a cavity in the vicinity of flavin, and (3) decreasing the glycosylation degree of enzymes. Among these approaches, altering the charge in the vicinity where the electron acceptor may be accessed will be most relevant.

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“…At the given conditions, the roGFP2-Orp1 sensor could not resolve these large differences in hydrogen peroxide formation since its turnover rates could not keep pace with elevated production. However, the fluorescent probe was successfully used to identify H 2 O 2 formation from variant N593C ( Brugger et al. , 2016 ), a rather strict dehydrogenase with substantially reduced oxygen reactivity (0.18 s −1 ), which underlines the exceptional sensitivity of roGFP2-Orp1.…”

Section: Discussionmentioning

confidence: 99%

Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1

Herzog

Borghi

Traxlmayr

et al. 2020

Protein Engineering, Design and Selection

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Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.

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Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity

Abstract: Pyranose oxidase (wtPOX) is turned into its equivalent active dehydrogenase by a single AA exchange (N593C-POx).

Cited by 7 publications

References 27 publications

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases

Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1

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