Biotechnology Letters

, Volume 41, Issue 1, pp 171–180 | Cite as

Enzyme-catalyzed regio-selective demethylation of papaverine by CYP105D1

  • Chen Shen
  • Wanli Zhao
  • Xuming LiuEmail author
  • Jihua LiuEmail author
Original Research Paper



To investigate the regio-selective demethylation of papaverine by CYP105D1 and develop a whole-cell biocatalytic system for the preparative synthesis of 6-O-demethyl-papaverine.


CYP105D1 from Streptomyces griseus ATCC 13273 was used for the regioselective demethylation of papaverine at C-6 using putidaredoxin reductase (PDR) and putidaredoxin (Pdx) as the electron transport system. The Km value of CYP105D1 towards papaverine was estimated to be 92.24 μM. Furthermore, a CYP105D1-based whole-cell system was established in E. coli BL21(DE3). The whole cell biotransformation condition was optimized as 25 °C, pH 7.5, 8 g (cell dry weight) L−1 whole cell biomass and 3% (v/v) PEG-200 as cosolvent. Under the optimal condition, the conversion yield of papaverine reached to 61.15% within 24 h.


The selective demethylation of papaverine by CYP105D1 was accomplished. The CYP105D1-based whole-cell biocatalyst has a potential used for the efficient synthesis of 6-O-demethyl-papaverine.


Biocatalysis Cytochrome P450 O-demethylation Papaverine Whole-cell biotransformation 



This work was supported by the Major Scientific and Technological Specialized Project for ‘New Drugs Development’ (No. 2012ZX09J12110-06B), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Supporting information

Supplementary Table 1—Primers used in this study.

Supplementary Table 2—Strains and plasmids used in this study.

Supplementary Table 3—HPLC conditions for analysis of papaverine biocatalytic profile.

Supplementary Fig. 1—Expression and purification of CYP105D1, Pdx and PDR.

Supplementary Fig. 2—The codon-optimized sequence of the gene encoding putidaredoxin reductase (PDR).

Supplementary Fig. 3—The codon-optimized sequence of the gene encoding putidaredoxin (Pdx).

Supplementary Fig. 4—1H NMR analysis spectrum of the product 6-O-demethylpapaverine (MeOD, 500 MHz).

Supplementary Fig. 5—13C NMR analysis spectrum of the product 6-O-demethylpapaverine (MeOD, 126 MHz).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2626_MOESM1_ESM.doc (1 mb)
Electronic supplementary material 1 (DOC 1065 kb)


  1. Awalt JK, Lam R, Kellam B, Graham B, Scammells PJ, Singer RD (2017) Utility of iron nanoparticles and a solution-phase iron species for the N-demethylation of alkaloids. Green Chem 19:2587–2594CrossRefGoogle Scholar
  2. Davila JC, Reddy CG, Davis PJ, Acosta D (1990) Toxicity assessment of papaverine hydrochloride and papaverine-derived metabolites in primary cultures of rat hepatocytes. Vitro Cell Dev Biol 26:515–524CrossRefGoogle Scholar
  3. Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Structure and chemistry of cytochrome P450. Chem Rev 105:2253–2277CrossRefGoogle Scholar
  4. Ewen KM et al (2009) Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450. J Biol Chem 284:28590–28598CrossRefGoogle Scholar
  5. Farrow SC, Facchini PJ (2015) Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe(2+)-dependent dioxygenase from opium poppy. FEBS Lett 589:2701–2706CrossRefGoogle Scholar
  6. Friedrich S, Hahn F (2015) Opportunities for enzyme catalysis in natural product chemistry. Tetrahedron 71:1473–1508CrossRefGoogle Scholar
  7. Grogan G (2011) Cytochromes P450: exploiting diversity and enabling application as biocatalysts. Curr Opin Chem Biol 15:241–248CrossRefGoogle Scholar
  8. Hagel JM, Facchini PJ (2013) Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol 54:647–672CrossRefGoogle Scholar
  9. Hlavica P (2009) Assembly of non-natural electron transfer conduits in the cytochrome P450 system: a critical assessment and update of artificial redox constructs amenable to exploitation in biotechnological areas. Biotechnol Adv 27:103–121CrossRefGoogle Scholar
  10. Huang H, Li L, Zhang H, Wei A (2017) Papaverine selectively inhibits human prostate cancer cell (PC-3) growth by inducing mitochondrial mediated apoptosis, cell cycle arrest and downregulation of NF-κB/PI3K/Akt signalling pathway. JBUON 22:112–118Google Scholar
  11. Jung E, Park BG, Ahsan MM, Kim J, Yun H, Choi KY, Kim BG (2016) Production of omega-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo. Appl Microbiol Biotechnol 100:10375–10384CrossRefGoogle Scholar
  12. Kaneda T, Shimizu K, Nakajyo S, Urakawa N (1998) The difference in the inhibitory mechanisms of papaverine on vascular and intestinal smooth muscles. Eur J Pharmacol 355:149–157CrossRefGoogle Scholar
  13. Marangoz AH, Kocacan SE, Him A, Kuruoglu E, Cokluk C, Marangoz C (2017) Proconvulsant effect of papaverine on penicillin-induced epileptiform activity in rats. Turk Neurosurg 28:479–482Google Scholar
  14. Mo J, Guo Y, Yang Y-S, Shen J-S, Jin G-Z, Zhen X (2007) Recent developments in studies of l-stepholidine and its analogs: chemistry, pharmacology and clinical implications. Curr Med Chem 14:2996–3002CrossRefGoogle Scholar
  15. Pandey BP, Lee N, Choi KY, Kim JN, Kim EJ, Kim BG (2014) Identification of the specific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680. Appl Microbiol Biotechnol 98:5009–5017CrossRefGoogle Scholar
  16. Rosazza JP, Kammer M, Youel L (1977) Microbial models of mammalian metabolism O-demethylations of papaverine. Xenobiotica 7:133–143CrossRefGoogle Scholar
  17. Taylor M, Lamb DC, Cannell R, Dawson M, Kelly SL (1999) Cytochrome P450105D1 (CYP105D1) from Streptomyces griseus: heterologous expression, activity, and activation effects of multiple xenobiotics. Biochem Biophys Res Commun 263:838–842CrossRefGoogle Scholar
  18. Taylor M, Lamb DC, Cannell RJ, Dawson MJ, Kelly SL (2000) Cofactor recycling with immobilized heterologous cytochrome P450 105D1 (CYP105D1). Biochem Biophys Res Commun 279:708–711CrossRefGoogle Scholar
  19. Trower MK, Lenstra R, Omer C, Buchholz SE, Sariaslani FS (1992) Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy (soyC) and ferredoxin soy (soyB) from Streptomyces griseus. Mol Microbiol 6:2125–2134CrossRefGoogle Scholar
  20. Ueno M, Yamashita M, Hashimoto M, Hino M, Fujie A (2005) Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24. J Biosci Bioeng 100:567–572CrossRefGoogle Scholar
  21. Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36CrossRefGoogle Scholar
  22. Zhang MX, Hu XH, Xu YH, Loh TP (2015) Selective Dealkylation of Alkyl Aryl Ethers. Asian J Org Chen 4:1047–1049CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese PharmacyChina Pharmaceutical UniversityNanjingChina
  2. 2.School of Life Science and TechnologyChina Pharmaceutical UniversityNanjingChina
  3. 3.State Key Laboratory of Natural MedicinesChina Pharmaceutical UniversityNanjingChina

Personalised recommendations