Applied Microbiology and Biotechnology

, Volume 97, Issue 4, pp 1625–1635 | Cite as

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

  • Marco Girhard
  • Florian Tieves
  • Evelyne Weber
  • Martha Sophia Smit
  • Vlada B. Urlacher
Biotechnologically relevant enzymes and proteins

Abstract

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (KM of 13.8 μM and kcat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.

Keywords

Candida apicola Cytochrome P450-diflavin reductase CPR P450 Heterologous expression 

Supplementary material

253_2012_4026_MOESM1_ESM.pdf (330 kb)
ESM 1(PDF 330 kb)

References

  1. Aigrain L, Pompon D, Truan G (2011) Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH-cytochrome P450 reductase. Biochem J 435:197–206CrossRefGoogle Scholar
  2. Arisawa A, Agematu H (2007) A modular approach to biotransformation using microbial cytochrome P450 monooxygenases. In: Schmid RD, Urlacher VB (eds) Modern biooxidation, 1st edn. Wiley-VCH, Weinheim, pp 177–192CrossRefGoogle Scholar
  3. Bernhardt R (1996) Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev Physiol Biochem Pharmacol 127:137–221CrossRefGoogle Scholar
  4. Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145CrossRefGoogle Scholar
  5. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefGoogle Scholar
  6. Craft DL, Madduri KM, Eshoo M, Wilson CR (2003) Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to alpha, omega-dicarboxylic acids. Appl Environ Microbiol 69:5983–5991CrossRefGoogle Scholar
  7. Fairhead M, Giannini S, Gillam EM, Gilardi G (2005) Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego. J Biol Inorg Chem 10:842–853CrossRefGoogle Scholar
  8. Gillam EM, Guo Z, Martin MV, Jenkins CM, Guengerich FP (1995) Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch Biochem Biophys 319:540–550CrossRefGoogle Scholar
  9. Girhard M, Schuster S, Dietrich M, Durre P, Urlacher VB (2007) Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase. Biochem Biophys Res Commun 362:114–119CrossRefGoogle Scholar
  10. Girhard M, Machida K, Itoh M, Schmid RD, Arisawa A, Urlacher VB (2009) Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system. Microb Cell Fact 8:36CrossRefGoogle Scholar
  11. Girhard M, Klaus T, Khatri Y, Bernhardt R, Urlacher VB (2010) Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis. Appl Microbiol Biotechnol 87:595–607CrossRefGoogle Scholar
  12. Guengerich FP, Martin MV, Sohl CD, Cheng Q (2009) Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat Protoc 4:1245–1251CrossRefGoogle Scholar
  13. Hajsig M (1958) Torulopsis apicola nov. spec., new isolated from bees. Antonie Van Leeuwenhoek 24:18–22CrossRefGoogle Scholar
  14. 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 124:172–181CrossRefGoogle Scholar
  15. Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems—biological variations of electron transport chains. Biochim Biophys Acta 1770:330–344CrossRefGoogle Scholar
  16. Kargel E, Menzel R, Honeck H, Vogel F, Bohmer A, Schunck WH (1996) Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum. Yeast 12:333–348CrossRefGoogle Scholar
  17. Khatri Y, Girhard M, Romankiewicz A, Ringle M, Hannemann F, Urlacher VB, Hutter MC, Bernhardt R (2010) Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56. Appl Microbiol Biotechnol 88:485–495CrossRefGoogle Scholar
  18. Kurtzman CP, Price NP, Ray KJ, Kuo TM (2010) Production of sophorolipid biosurfactants by multiple species of the Starmerella (Candida) bombicola yeast clade. FEMS Microbiol Lett 311:140–146CrossRefGoogle Scholar
  19. Lamb DC, Warrilow AG, Venkateswarlu K, Kelly DE, Kelly SL (2001) Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase. Biochem Biophys Res Commun 286:48–54CrossRefGoogle Scholar
  20. Lottermoser K, Schunck WH, Asperger O (1996) Cytochromes P450 of the sophorose lipid-producing yeast Candida apicola: heterogeneity and polymerase chain reaction-mediated cloning of two genes. Yeast 12:565–575CrossRefGoogle Scholar
  21. Maurer SC, Schulze H, Schmid RD, Urlacher VB (2003) Immobilisation of P450 BM-3 and an NADP+ cofactor recycling system: towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv Synth Catal 345:802–810CrossRefGoogle Scholar
  22. Mishra RN, Singla-Pareek SL, Nair S, Sopory SK, Reddy MK (2002) Directional genome walking using PCR. Biotechniques 33:830–834Google Scholar
  23. Munro AW, Noble MA, Robledo L, Daff SN, Chapman SK (2001) Determination of the redox properties of human NADPH-cytochrome P450 reductase. Biochemistry 40:1956–1963CrossRefGoogle Scholar
  24. Nelson DR (2011) Progress in tracing the evolutionary paths of cytochrome P450. Biochim Biophys Acta 1814:14–18CrossRefGoogle Scholar
  25. Omura T, Sato R (1964a) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378Google Scholar
  26. Omura T, Sato R (1964b) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J Biol Chem 239:2379–2385Google Scholar
  27. Park HG, Lim YR, Eun CY, Han S, Han JS, Cho KS, Chun YJ, Kim D (2010) Candida albicans NADPH-P450 reductase: expression, purification, and characterization of recombinant protein. Biochem Biophys Res Commun 396:534–538CrossRefGoogle Scholar
  28. Pritchard MP, Ossetian R, Li DN, Henderson CJ, Burchell B, Wolf CR, Friedberg T (1997) A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1. Arch Biochem Biophys 345:342–354CrossRefGoogle Scholar
  29. Purnapatre K, Khattar SK, Saini KS (2008) Cytochrome P450s in the development of target-based anticancer drugs. Cancer Lett 259:1–15CrossRefGoogle Scholar
  30. Rupasinghe SG, Duan H, Frericks Schmidt HL, Berthold DA, Rienstra CM, Schuler MA (2007) High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy. Biochim Biophys Acta 1768:3061–3070CrossRefGoogle Scholar
  31. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  32. Sevrioukova I, Truan G, Peterson JA (1996) The flavoprotein domain of P450BM-3: expression, purification, and properties of the flavin adenine dinucleotide- and flavin mononucleotide-binding subdomains. Biochemistry 35:7528–7535CrossRefGoogle Scholar
  33. Sevrioukova I, Truan G, Peterson JA (1997) Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains. Arch Biochem Biophys 340:231–238CrossRefGoogle Scholar
  34. Sutter TR, Sanglard D, Loper JC (1990) Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins. J Biol Chem 265:16428–16436Google Scholar
  35. Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36Google Scholar
  36. Van Bogaert IN, Develter D, Soetaert W, Vandamme EJ (2007) Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) from Candida bombicola. FEMS Yeast Res 7:922–928CrossRefGoogle Scholar
  37. Van Bogaert IN, De Mey M, Develter D, Soetaert W, Vandamme EJ (2009) Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola. FEMS Yeast Res 9:87–94CrossRefGoogle Scholar
  38. van den Brink HJ, van Zeijl CM, Brons JF, van den Hondel CA, van Gorcom RF (1995) Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger. DNA Cell Biol 14:719–729CrossRefGoogle Scholar
  39. Wang M, Roberts DL, Paschke R, Shea TM, Masters BS, Kim JJ (1997) Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc Natl Acad Sci U S A 94:8411–8416CrossRefGoogle Scholar
  40. Weber L, Stach J, Haufe G, Hommel R, Kleber HP (1990) Elucidation of the structure of an unusual cyclic glycolipid from Torulopsis apicola. Carbohydr Res 206:13–19CrossRefGoogle Scholar
  41. Yun CH, Yim SK, Kim DH, Ahn T (2006) Functional expression of human cytochrome P450 enzymes in Escherichia coli. Curr Drug Metab 7:411–429CrossRefGoogle Scholar
  42. Zimmer T, Ohkuma M, Ohta A, Takagi M, Schunck WH (1996) The CYP52 multigene family of Candida maltosa encodes functionally diverse n-alkane-inducible cytochromes P450. Biochem Biophys Res Commun 224:784–789CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Marco Girhard
    • 1
  • Florian Tieves
    • 1
  • Evelyne Weber
    • 2
  • Martha Sophia Smit
    • 3
  • Vlada B. Urlacher
    • 1
  1. 1.Institute of BiochemistryHeinrich-Heine University DüsseldorfDüsseldorfGermany
  2. 2.Institute of Technical BiochemistryUniversität StuttgartStuttgartGermany
  3. 3.Department of Microbial, Biochemical and Food BiotechnologyUniversity of the Free StateBloemfonteinSouth Africa

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