Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450

Kwon-Young Choi,Hyung-Yeon Park,Changmin Sung,Bishnu Prasad Pandey,Byung-Gee Kim,Hyungdon Yun,Eunok Jung,Byeo-Ri An,Da-Hye Jung

doi:10.1186/1475-2859-11-81

Kwon-Young Choi, Hyung-Yeon Park + Show 7 more

Open Access

https://doi.org/10.1186/1475-2859-11-81

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Abstract

A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.

Highlights

Cytochrome P450 monooxygenases (CYPs) are a superfamily of heme-thiolate containing enzymes that catalyze a variety of chemical reactions in regio/stereo-selective manners [1]
The class III cytochrome P450 system consists of a self-sufficient CYP, in which the heme domain is fused with a P450 reductase domain and encoded in a single polypeptide [9,10]
Fusion-mediated self-sufficient construction of CYP105D7 with the reductase domain of CYP102D1 A recent study described the functional expression of a self-sufficient P450, CYP102D1, from S. avermitilis in E. coli [15]

Summary

Introduction

Cytochrome P450 monooxygenases (CYPs) are a superfamily of heme-thiolate containing enzymes that catalyze a variety of chemical reactions in regio/stereo-selective manners [1]. Various types of reactions catalyzed by CYP have been identified; generally, these enzymes act as monooxygenases that catalyze the introduction of an oxygen molecule into the substrate whereby one atom of molecular oxygen is incorporated into the product [2]. The class III cytochrome P450 system consists of a self-sufficient CYP, in which the heme domain is fused with a P450 reductase domain and encoded in a single polypeptide [9,10]. The knowledge that self-sufficient P450s exhibit the highest turnover frequency due to the higher possibility of contact between the heme and reductase domains, and the induction of intra-molecular electron transfer, have prompted several attempts to make an artificial selfsufficient system by fusing redox proteins. NCIMB 9784 to increase catalytic efficiency [19]

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