Volume 13, Issue 12 1800088

Research Article

Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

Tomoaki Haga

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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Hidehiko Hirakawa

Corresponding Author

hirakawa.hidehiko.ge@u.tsukuba.ac.jp

orcid.org/0000-0003-0623-8573

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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Teruyuki Nagamune

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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Tomoaki Haga

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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Hidehiko Hirakawa

Corresponding Author

hirakawa.hidehiko.ge@u.tsukuba.ac.jp

orcid.org/0000-0003-0623-8573

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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Teruyuki Nagamune

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113‐8656, Japan

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First published: 24 July 2018

https://doi.org/10.1002/biot.201800088

Citations: 3

Present address: Faculty of Life and Environmental Sciences, University of Tsukuba, 1‐1‐1 Tennodai, Tsukuba, Ibaraki 305‐8572, Japan

Abstract

Most bacterial cytochrome P450 monooxygenases (P450s) do not work alone because their active species is generated by two electrons supplied through two separate auxiliary proteins. Artificial “self‐sufficient” P450s, in which one molecule each of the two auxiliary proteins is arranged close to the P450s, have been developed but have not achieved the maximum catalytic turnover numbers of the P450s. In this study, the Pseudomonas putida P450 (P450cam) is assembled with multiple molecules of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), by fusion to a heterotrimeric protein. In the assembled P450cam containing one PdX and one PdR, kinetic analysis reveales that the catalytic cycle of P450cam is suspended twice awaiting the reduction of PdX by PdR. An increase in the number of PdR molecules stimulated the PdX reduction process. Assembly with two PdXs allows one PdX to be reduced during the binding of the other PdX to P450cam for the first electron transfer, eliminating one waiting step. Finally, P450cam assembled with two PdXs and three PdRs showes 92% of the maximum activity of free P450cam. Therefore, assembly with multiple molecules of auxiliary proteins will facilitate in vitro biotechnological applications of the P450s.

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Table S1. Potassium ferricyanide reduction activities of PdR‐fused proteins.

Table S2. NADH consumption rates of free P450cam in the presence of PdX‐fused proteins.

Table S3. NADH consumption rates of P450cam‐fused proteins.

Table S4. Status of PdX^ox in PUPPET^[a].

Table S5. Kinetic constants^[a] and binding status of PdX^ox in cytochrome c reduction reaction.

Table S6. NADH consumption rates of mixtures of P450cam and a complex containing PdR and PdX.

Table S7. Equations for estimation of the binding status of PdX^ox in spectral analysis of PUPPET.

Table S8. Estimated constant values.

Table S9. Equations for estimation of the binding status of PdX^ox in cytochrome c assay of PUPPET.

Figure S1. Component proteins of PUPPETs. (A) Protein constructs and (B) SDS‐PAGE analysis of the component proteins.

Figure S2. UV‐vis spectra of fusion proteins.

Figure S3. Preparation of PUPPET.

Figure S4. Effect of incubation time and temperature on the formation of PUPPET with intersubunit disulfide bonds.

Figure S5. UV‐vis spectra of PUPPET.

Figure S6. SDS‐PAGE and UV‐vis spectral analyses of protein complexes lacking P450cam.

Figure S7. Cytochrome c reduction activities of the protein complexes.

Figure S8. GC analysis of the monooxygenase reaction of PUPPET.

Figure S9. Relationship between initial NADH consumption rate and PdX concentration in the presence of P450cam.

Figure S10. Spectral changes of P450cam induced by the substrate and oxidized PdX.

Figure S11. Characterization of P450cam‐PCNA3R112C/T180C‐PdX.

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Volume13, Issue12

Special Issue: Biomimetic and Bioinspired Biotechnology

December 2018

1800088

Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

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