title: Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.
abstract: Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.
authors.affiliation: Faculty of Agriculture, Kyushu University, 6-10-(1) Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: m_hatakeyama@agr.kyushu-u.ac.jp.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Hatakeyama
authors.affiliation: Faculty of Agriculture, Kyushu University, 6-10-(1) Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: tkitaoka@agr.kyushu-u.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kitaoka
authors.affiliation: Faculty of Agriculture, Kyushu University, 6-10-(1) Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: ichinose@agr.kyushu-u.ac.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D015262:Xenobiotics; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Alternative electron transfer pathways; Chimerization; Cytochrome b(5); Escherichia coli; Fungal cytochrome P450; Heterologous expression