Monoterpene hydroxylation with an artificial self-sufficient P450 utilizing a P450SMO reductase domain for the electron transfer

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2015-06, Vol.116, p.78-82
• Main Authors: Luan, Zheng-Jiao, Yin, Yue-Cai, Li, Ai-Tao, Yu, Hui-Lei, Xu, Jian-He
• Format: Article
• Language: English
• Subjects: Electron transfer; Monoterpene hydroxylation; Protein engineering; Self-sufficient P450s
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2015.02.006

We constructed an artificial self-sufficient P450-type monoterpene hydroxylase by connecting the P450SMO reductase domain and the P450cam (Y96F/V247L) domain together with a linker region (G4S)4. The resultant chimeric P450 enzyme could catalyze the hydroxylation of (−)-limonene and α-pinene as well as camphor, which were all inactive for the natural fusion protein P450SMO. Co-expression of the fused P450 with a glucose dehydrogenase (GDH) improved the (−)-limonene conversion as sufficient NADPH was regenerated in the system with glucose as a cosubstrate. This work illustrated that P450SMO reductase can act well as an electron donor partner of P450s and may be used for fusion with heterogeneous P450 domains to elucidate the catalytic function of other unknown P450s. •The reductase domain of P450SMO can be used as the electron donor partner for Class I P450.•The linker (G4S)4 exhibiting high flexibility showed high catalytic activity.•The fusion enzyme could catalyze the hydroxylation of (−)-limonene, α-pinene as well as camphor.•Co-expression of GDH with the artificial P450 can supply enough NADPH for the hydroxylation. Cytochrome P450SMO from Rhodococcus sp. ECU0066 is a natural self-sufficient P450 monooxygenase, consisting of a heme domain, a flavin-reductase domain containing FMN and NADPH binding sites, and a [Fe2S2] ferredoxin domain. P450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor. The variant P450cam (Y96F/V247L) was reported for the oxidation of monoterpene by protein engineering. In this work, we constructed an artificial self-sufficient P450-type monoterpene hydroxylase by connecting the P450SMO reductase domain and the P450cam (Y96F/V247L) domain together with a linker region (G4S)4. The resultant chimeric P450 enzyme could catalyze the hydroxylation of (−)-limonene and α-pinene as well as camphor, which were all inactive for the natural fusion protein P450SMO. Co-expression of the fused P450 with a glucose dehydrogenase (GDH) improved the (−)-limonene conversion as sufficient NADPH was regenerated in the system with glucose as a cosubstrate. This work illustrated that P450SMO reductase might act as an electron donor partner of P450s and might be used for fusion with heterogeneous P450 domains to elucidate the catalytic function of other unknown P450s.