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Catalytically self-sufficient CYP116B5: domain switch for improved peroxygenase activity
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  • Danilo Correddu,
  • Gianluca Catucci,
  • Daniele Giuriato,
  • Giovanna Di Nardo,
  • Alberto Ciaramella,
  • Gianfranco Gilardi
Danilo Correddu
University of Turin
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Gianluca Catucci
University of Turin
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Daniele Giuriato
University of Turin
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Giovanna Di Nardo
University of Turin
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Alberto Ciaramella
University of Turin
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Gianfranco Gilardi
University of Turin

Corresponding Author:[email protected]

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Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions towards a wide range of organic compounds without the need of a separate reductase partner. However, these P450s are often unstable in solution and their activity is limited to short reaction time. As the isolated heme domain of CYP116B5 has been shown to work as a peroxygenase with H2O2 without the need for expensive NAD(P)H, in this work protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) that is able to produce H2O2 with a controlled and continuous release in time. The full-length form enzyme (CYP116B5-fl) is expressed and characterized for the first time, allowing a detailed comparison to both the isolated heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2O2 (CYP116B5-hd) and sarcosine (CYP116B5-SOX) as direct or indirect source of electrons. CYP116B5-SOX outperforms CYP116B5-fl by 10 folds and CYP116B5-hd by 3 folds, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.
11 Dec 2022Submitted to Biotechnology Journal
14 Dec 2022Submission Checks Completed
14 Dec 2022Assigned to Editor
22 Dec 2022Reviewer(s) Assigned
11 Jan 2023Review(s) Completed, Editorial Evaluation Pending
11 Jan 2023Editorial Decision: Revise Minor
24 Jan 20231st Revision Received
25 Jan 2023Submission Checks Completed
25 Jan 2023Assigned to Editor
02 Feb 2023Reviewer(s) Assigned
19 Feb 2023Review(s) Completed, Editorial Evaluation Pending
21 Feb 2023Editorial Decision: Accept