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Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents

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The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).

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  1. Anzenbacherova E, Bec N, Anzenbacher P et al (2000) Flexibility and stability of the structure of cytochromes P450 3A4 and BM-3. Eur J Biochem 267(10):2916–2920

  2. Appel D, Lutz-Wahl S, Fischer P et al (2001) A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J Biotechnol 88(2):167–171

  3. Boddupalli SS, Estabrook RW, Peterson JA (1990) Fatty acid monooxygenation by cytochrome P-450BM-3. J Biol Chem 265(8):4233–4239

  4. Boddupalli SS, Oster T, Estabrook RW et al (1992) Reconstitution of the fatty-acid hydroxylation function of cytochrome-p-450bm-3 utilizing its individual recombinant hemoprotein and flavoprotein domains. J Biol Chem 267(15):10375–10380

  5. Carmichael AB, Wong LL (2001) Protein engineering of Bacillus megaterium CYP102.The oxidation of polycyclic aromatic hydrocarbons. Eur J Biochem 268(10):3117–3125

  6. Gustafsson MC, Roitel O, Marshall KR et al (2004) Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium. Biochemistry 43(18):5474–5487

  7. Jamakhandi AP, Jeffus BC, Dass VR et al (2005) Thermal inactivation of the reductase domain of cytochrome P450 BM3. Arch Biochem Biophys 439(2):165–174

  8. Kubo T, Peters MW, Meinhold P et al (2006) Enantioselective epoxidation of terminal alkenes to(R)- and (S)-epoxides by engineered cytochromes P450BM-3. Chemistry 12(4):1216–1220

  9. Kunst F, Ogasawara N, Moszer I et al (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390(6657):249–256

  10. Laemmli UK, Beguin F, Gujerkel G (1970) A factor preventing major head protein of bacteriophage T4 from random aggregation. J Mol Biol 47(1):69–85

  11. Lentz O, Urlacher V, Schmid RD (2004) Substrate specificity of native and mutated cytochrome P450 (CYP102A3) from Bacillus subtilis. J Biotechnol 108(1):41–49

  12. Maurer SC, Schulze H, Schmid RD et al (2003) Immobilisation of P450BM-3 and an NADP(+) cofactor recycling system: towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv Synth Catal 345(6–7):802–810

  13. Maurer SC, Kuhnel K, Kaysser LA et al (2005) Catalytic hydroxylation in biphasic systems using CYP102A1 mutants. Adv Synth Catal 347(7–8):1090–1098

  14. McLean KJ, Scrutton NS, Munro AW (2003) Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. Biochem J 372:317–327

  15. Minoletti C, Dijols S, Dansette PM et al (1999) Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18. Biochemistry 38(24):7828–7836

  16. Munro AW, Lindsay JG, Coggins JR et al (1996) Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3. Biochim Biophys Acta 1296(2):127–137

  17. Narhi LO, Fulco AJ (1986) Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem 261(16):7160–7169

  18. Neeli R, Girvan HM, Lawrence A et al (2005) The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase. FEBS Lett 579(25):5582–5588

  19. Omura T, Sato R (1964) Carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239(7):2370–2378

  20. Parikh A, Josephy PD, Guengerich FP (1999) Selection and characterization of human cytochrome 1A2 mutants with altered catalytic properties. Biochemistry 38(17):5283–5289

  21. Schwaneberg U, Schmidt-Dannert C, Schmitt J et al (1999) A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal Biochem 269(2):359–366

  22. Sibbesen O, De Voss JJ, Montellano PR (1996) Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein. Construction of a self-sufficient Escherichia coli catalytic system. J Biol Chem 271(37):22462–22469

  23. Tishkov VI, Galkin AG, Fedorchuk VV et al (1999) Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases. Biotechnol Bioeng 64(2):187–193

  24. Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24(7):324–330

  25. Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1(6):3003.1–3003.3009

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We thank Professor Dr. Rolf D. Schmid for his support and helpful discussions.

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Correspondence to Vlada B. Urlacher.

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Eiben, S., Bartelmäs, H. & Urlacher, V.B. Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents. Appl Microbiol Biotechnol 75, 1055–1061 (2007). https://doi.org/10.1007/s00253-007-0922-z

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  • Cytochrome P450 monooxygenase
  • Thermostability
  • Chimera
  • Self-sufficient