Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins

Ketaki D. Belsare; Anna Joëlle Ruff; Ronny Martinez; Ulrich Schwaneberg

doi:10.1515/hsz-2020-0134

Article

Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins

Ketaki D. Belsare , Anna Joëlle Ruff , Ronny Martinez and Ulrich Schwaneberg

Published/Copyright: July 16, 2020

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From the journal Biological Chemistry Volume 401 Issue 11

Abstract

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA–CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA–CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

Keywords: cytochrome P450; directed evolution; fusion protein; intermolecular interaction; protein linkers

Corresponding author: Ronny Martinez, Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, Aachen, D-52074, Germany; and Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, 1720010, Chile, E-mail: remartinez@userena.cl

Funding source: Arbeitsgemeinschaft industrieller Forschungsvereinigungen (AiF)

Award Identifier / Grant number: F 519F

Acknowledgments

Our work was supported by the Arbeitsgemeinschaft industrieller Forschungsvereinigungen (AiF) Otto von Guericke e. V. [grant number: F 519F].

Author contribution: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Our work was supported by the Arbeitsgemeinschaft industrieller Forschungsvereinigungen (AiF) Otto von Guericke e. V. [grant number: F 519F].
Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2020-0134).

Received: 2020-02-12

Accepted: 2020-05-25

Published Online: 2020-07-16

Published in Print: 2020-10-25

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Supplementary Material

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Keywords for this article

cytochrome P450; directed evolution; fusion protein; intermolecular interaction; protein linkers