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Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

  • Original Article
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Lipids

Abstract

Molecular evolution is a powerful tool for improving or changing activities of enzymes for their use in biotechnological processes. Cytochromes P450 are highly interesting enzymes for biotechnological purposes because they are able to hydroxylate a broad variety of substrates with high regio- and stereoselectivity. One promising steroid hydroxylating cytochrome P450 for biotechnological applications is CYP106A2 from Bacillus megaterium ATCC 13368. It is one of a few known bacterial cytochromes P450 able to transform steroids such as progesterone and 11-deoxycortisol. CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase. We developed a simple screening assay for this system and performed random mutagenesis of CYP106A2, yielding variants with improved 11-deoxycortisol and progesterone hydroxylation activity. After two generations of directed evolution, we were able to improve the k cat/K m of the 11-deoxycortisol hydroxylation by a factor of more than four. At the same time progesterone conversion was improved about 1.4-fold. Mapping the mutations identified in catalytically improved CYP106A2 variants into the structure of a CYP106A2 model suggests that these mutations influence the mobility of the F/G loop, and the interaction with the redox partner adrenodoxin. The results show the evolution of a soluble steroid hydroxylase as a potential new catalyst for the production of steroidogenic compounds.

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Abbreviations

AdR:

Adrenodoxin reductase

Adx:

Adrenodoxin

CYP:

Cytochrome P450

DOC:

Deoxycorticosterone

HPLC:

High performance liquid chromatography

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

P:

Progesterone

RFU.:

Relative fluorescence units

RSS:

11-deoxycortisol

WT:

Wild type

Deoxycorticosterone:

4-Pregnene-21-ol-3, 20-dione

11-Deoxycortisol:

4-Pregnene-17, 21-diol-3, 20-dione

Progesterone:

4-Pregnene-3, 20-dione

References

  1. Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145

    Article  PubMed  CAS  Google Scholar 

  2. Isin EM, Guengerich FP (2007) Complex reactions catalyzed by cytochrome P450 enzymes. Biochim Biophys Acta 1770:314–329

    PubMed  CAS  Google Scholar 

  3. Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta 1770:330–344

    PubMed  CAS  Google Scholar 

  4. Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS (2003) Microbial conversion of steroid compounds: recent developments. Enzyme Microb Technol 32:688–705

    Article  CAS  Google Scholar 

  5. Chefson A, Auclair K (2006) Progress towards the easier use of P450 enzymes. Mol Biosyst 2:462–469

    Article  PubMed  CAS  Google Scholar 

  6. Mahato SB, Garai S (1997) Advances in microbial steroid biotransformation. Steroids 62:332–345

    Article  PubMed  CAS  Google Scholar 

  7. Jekkel A, Ilkóy É, Horváth G, Pallagi I, Süto J, Ambrus G (1998) Microbial hydroxylation of 13β-ethyl-4-gonene-3, 17-dione. J Mol Catal B Enzym 5:385–387

    Article  CAS  Google Scholar 

  8. Orencia MC, Yoon JS, Ness JE, Stemmer WP, Stevens RC (2001) Predicting the emergence of antibiotic resistance by directed evolution and structural analysis. Nat Struct Biol 8:238–242

    Article  PubMed  CAS  Google Scholar 

  9. Hart DJ, Tarendeau F (2006) Combinatorial library approaches for improving soluble protein expression in Escherichia coli. Acta Crystallogr D Biol Crystallogr 62:19–26

    Article  PubMed  Google Scholar 

  10. Appel D, Schmid RD, Dragan CA, Bureik M, Urlacher VB (2005) A fluorimetric assay for cortisol. Anal Bioanal Chem 383:182–186

    Article  PubMed  CAS  Google Scholar 

  11. Hannemann F, Virus C, Bernhardt R (2006) Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases. J. Biotechnol 124:172–181

    Article  PubMed  CAS  Google Scholar 

  12. Berg A, Gustafsson JA, Ingelman-Sundberg M (1976) Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium. J Biol Chem 251:2831–2838

    PubMed  CAS  Google Scholar 

  13. Lisurek M, Kang MJ, Hartmann RW, Bernhardt R (2004) Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry. Biochem Biophys Res Commun 319:677–682

    Article  PubMed  CAS  Google Scholar 

  14. Virus C, Lisurek M, Simgen B, Hannemann F, Bernhardt R (2006) Function and engineering of the 15beta-hydroxylase CYP106A2. Biochem Soc Trans 34:1215–1218

    Article  PubMed  CAS  Google Scholar 

  15. Berg A, Ingelman-Sundberg M, Gustafsson JA (1979) Purification and characterization of cytochrome P-450meg. J Biol Chem 254:5264–5271

    PubMed  CAS  Google Scholar 

  16. Simgen B, Contzen J, Schwarzer R, Bernhardt R, Jung C (2000) Substrate binding to 15beta-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration. Biochem Biophys Res Commun 269:737–742

    Article  PubMed  CAS  Google Scholar 

  17. Sagara Y, Wada A, Takata Y, Waterman MR, Sekimizu K, Horiuchi T (1993) Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization. Biol Pharm Bull 16:627–630

    PubMed  CAS  Google Scholar 

  18. Uhlmann H, Beckert V, Schwarz D, Bernhardt R (1992) Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of/2 Fe-2S/cluster ligands. Biochem Biophys Res Commun 188:1131–1138

    Article  PubMed  CAS  Google Scholar 

  19. Lisurek M, Simgen B, Antes I, Bernhardt R (2008) Theoretical and experimental evaluation of a CYP106A2 low homology model and production of mutants with changed activity and selectivity of hydroxylation. Chembiochem 9:1439–1449

    Article  PubMed  CAS  Google Scholar 

  20. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  PubMed  CAS  Google Scholar 

  21. Volin P (1995) High-performance liquid chromatographic analysis of corticosteroids. J Chromatogr B 671:319–340

    Article  CAS  Google Scholar 

  22. Shimada K, Mitamura K, Higashi T (2001) Gas chromatography and high-performance liquid chromatography of natural steroids. J Chromatogr A 935:141–172

    Article  PubMed  CAS  Google Scholar 

  23. Lewis JG, Manley L, Whitlow JC, Elder PA (1992) Production of a monoclonal antibody to cortisol: application to a direct enzyme-linked immunosorbent assay of plasma. Steroids 57:82–85

    Article  PubMed  CAS  Google Scholar 

  24. Mirasoli M, Deo SK, Lewis JC, Roda A, Daunert S (2002) Bioluminescence immunoassay for cortisol using recombinant aequorin as a label. Anal Biochem 306:204–211

    Article  PubMed  CAS  Google Scholar 

  25. Goldzieher JW, Bodenchuk JM, Nolan P (1954) Fluorescence reactions of steroids. Anal Chem 26:853–856

    Article  CAS  Google Scholar 

  26. Dunn AR, Dmochowski IJ, Bilwes AM, Gray HB, Crane BR (2001) Probing the open state of cytochrome P450cam with ruthenium-linker substrates. Proc Natl Acad Sci USA 98:12420–12425

    Article  PubMed  CAS  Google Scholar 

  27. Park SY, Yamane K, Adachi S, Shiro Y, Weiss KE, Maves SA, Sligar SG (2002) Thermophilic cytochrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties. J Inorg Biochem 91:491–501

    Article  PubMed  CAS  Google Scholar 

  28. Geren LM, O’Brien P, Stonehuerner J, Millett F (1984) Identification of specific carboxylate groups on adrenodoxin that are involved in the interaction with adrenodoxin reductase. J Biol Chem 259:2155–2160

    PubMed  CAS  Google Scholar 

  29. Geren L, Tuls J, O’Brien P, Millett F, Peterson JA (1986) The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase. J Biol Chem 261:15491–15495

    PubMed  CAS  Google Scholar 

  30. Bernhardt R, Makower A, Jänig G-R, Ruckpaul K (1984) Selective chemical modification of a functionally linked lysine in cytochrome P-450LM2. Biochim Biophys Acta 785:186–190

    PubMed  CAS  Google Scholar 

  31. Bernhardt R, Pommerening K, Ruckpaul K (1987) Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450LM2. Biochem Int 14:823–832

    PubMed  CAS  Google Scholar 

  32. Coghlan VM, Vickery LE (1991) Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc. J Biol Chem 266:18606–18612

    PubMed  CAS  Google Scholar 

  33. Stayton PS, Sligar SG (1990) The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling. Biochemistry 29:7381–7386

    Article  PubMed  CAS  Google Scholar 

  34. Wada A, Waterman MR (1992) Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding. J Biol Chem 267:22877–22882

    PubMed  CAS  Google Scholar 

  35. Pochapsky TC, Lyons TA, Kazanis S, Arakaki T, Ratnaswamy G (1996) A structure-based model for cytochrome P450cam-putidaredoxin interactions. Biochimie 78:723–733

    Article  PubMed  CAS  Google Scholar 

  36. Koga H, Sagara Y, Yaoi T, Tsujimura M, Nakamura K, Sekimizu K, Makino R, Shimada H, Ishimura Y, Yura K et al (1993) Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin. FEBS Lett 331:109–113

    Article  PubMed  CAS  Google Scholar 

  37. Nakamura K, Horiuchi T, Yasukochi T, Sekimizu K, Hara T, Sagara Y (1994) Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin. Biochim Biophys Acta 20:40–48

    Google Scholar 

  38. Unno M, Shimada H, Toba Y, Makino R, Ishimura Y (1996) Role of Arg112 of cytochrome p450cam in the electron transfer from reduced putidaredoxin. Analyses with site-directed mutants. J Biol Chem 271:17869–17874

    Article  PubMed  CAS  Google Scholar 

  39. Roitberg A, Holden M, Mayhew M, Kurnikov IV, Beratan DN, Vilker V (1998) Binding and electron transfer between putidaredoxin and cytochrome P450cam. Theory and experiments. J Am Chem Soc 120:8927–8932

    Article  CAS  Google Scholar 

  40. Stayton PS, Poulos TL, Sligar SG (1989) Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex. Biochemistry 28:8201–8205

    Article  PubMed  CAS  Google Scholar 

  41. Bichet A, Hannemann F, Rekowski M, Bernhardt R (2007) A new application of the yeast two-hybrid system in protein engineering. Protein Eng Des Sel 20:117–123

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Wolfgang Reinle for excellent purification of adrenodoxin reductase and adrenodoxin. A grant of the Fonds der Chemischen Industrie to R.B. is acknowledged.

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Authors and Affiliations

  1. Naturwissenschaftlich-Technische Fakultät III, Institut für Biochemie, Universität des Saarlandes, Postfach 151150, 66041, Saarbrücken, Germany

    Cornelia Virus & Rita Bernhardt

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  1. Cornelia Virus
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  2. Rita Bernhardt
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Correspondence to Rita Bernhardt.

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Virus, C., Bernhardt, R. Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening. Lipids 43, 1133–1141 (2008). https://doi.org/10.1007/s11745-008-3236-8

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  • DOI: https://doi.org/10.1007/s11745-008-3236-8

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