In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins
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The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.
KeywordsP450 hydroxylase Cyclosporin A Hair-stimulating agent Redox partner proteins Synechococcus elongatus
This work was supported by National Natural Science Foundation of China under Grant Nos. NSFC 31422002 (S. Li) and 31300663 (D. Han), the Natural Science Foundation of Shandong Province, China (No. ZR2016CQ05) (L. Ma), and the National Research Foundation (NRF) of Korea (No. NRF-2014R1A2A1A11052236) (E.-S. Kim). We are grateful to the financial support from the Applied Basic Research Programs of Science and Technology of Qingdao (14-2-4-10-jch). We thank Prof. Xuefeng Lu at Qingdao Institute of Bioenergy and Biotechnology, Chinese Academy of Sciences, for providing us with the S. elongatus PCC7942 genome DNA.
- 4.Chun YJ, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao B, Kelly SL, Waterman MR, Lamb DC, Guengerich FP (2007) Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2). J Biol Chem 282:17486–17500CrossRefPubMedGoogle Scholar
- 6.Kim SN AH, Lee CW, Lee MH, Kim JH, Kim JI, Kim SJ, Cho HS, Lee HS, Kim HJ (2004) The use of nonimmunosuppressive [γ-hydroxy-N-methyl-l-leucine4] cyclosporin derivatives for treating hair loss. Patent DE-1392224, WO02/092033 A1Google Scholar
- 11.Ma L, Du L, Chen H, Sun Y, Huang S, Zheng X, Kim ES, Li S (2015) Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system. Appl Environ Microbiol 81:6268–6275CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Nelson DR (2009) The cytochrome P450 homepage. Hum Genom 4:59–65Google Scholar