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

Author:

Belsare Ketaki D.12,Ruff Anna Joëlle1,Martinez Ronny13,Schwaneberg Ulrich14

Affiliation:

1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, Aachen, D-52074, Germany

2. University of California at San Francisco, 555 Mission Bay Blvd South, San Francisco, 94158, CA, USA

3. Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena, 1720010, Chile

4. DWI – Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, Aachen, D-52074, Germany

Abstract

AbstractCytochrome 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.

Funder

Arbeitsgemeinschaft industrieller Forschungsvereinigungen

Publisher

Walter de Gruyter GmbH

Subject

Clinical Biochemistry,Molecular Biology,Biochemistry

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