Skip to main content
SpringerLink
Log in

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis

  • Original Article
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker ν4 at 1373 cm−1 (indicating ferric P450 haem) and the splitting of the spin-state marker ν3 into two components (1503 cm−1 and 1488 cm−1), indicating cytochrome P450 BioI to be a mixture of highand low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (K d=4.18±0.26 μM) and pentadecanoic acid (K d=3.58±0.54 μM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (K d<1 μM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the ω-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BIM:

n-butylimidazole

BioI:

Bacillus subtilis cytochrome P450 BioI

CPR:

scytochrome P450 reductase

ESIMS:

electrospray ionization mass spectrometry

FLD:

Escherichia coli flavodoxin

FLDR:

E. coli flavodoxin NADP+ oxidoreductase

Im:

imidazole

ImC12:

12-imidazolyldodecanoic acid

IPTG:

isopropyl-β,D-thiogalactopyranoside

P450:

cytochrome P450-dependent monooxygenase

1-PIM:

1-phenylimidazole

References

  1. Zempleni J, Mock DM (1999) J Nutr Biochem 3:128–138

    Article  Google Scholar 

  2. Wallace JC, Jitrapakdee S, Chapman-Smith A (1998) Int J Biochem Cell Biol 30:1–5

    Article  PubMed  CAS  Google Scholar 

  3. Baldet P, Alban C, Douce R (1997) Methods Enzymol 279:327–339

    Article  PubMed  CAS  Google Scholar 

  4. Bui BTS, Marquet A (1997) Methods Enzymol 279:356–362

    Article  PubMed  CAS  Google Scholar 

  5. Gloeckler R, Ohsawa I, Speck D, Ledoux C, Bernard S, Zinsius M, Villeval D, Kisou T, Kamogawa K, Lemoine Y (1990) Gene 87:63–70

    Article  PubMed  CAS  Google Scholar 

  6. Ploux O, Soularue P, Marquet A, Gloeckler R, Lemoine Y (1992) Biochem J 287:685–690

    PubMed  CAS  Google Scholar 

  7. Lemoine Y, Wach A, Jeltsch JM (1996) Mol Microbiol 19:645–647

    Article  PubMed  CAS  Google Scholar 

  8. Bower S, Perkins JB, Yocum RR, Howitt CL, Rahaim P, Pero J (1996) J Bacteriol 178:4122–4130

    PubMed  CAS  Google Scholar 

  9. Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Estabrook RW, Feyereisen R, Gonzalez FJ, Coon MJ, Gunsalus IC, Gotoh O, Okuda K, Nebert DW (1993) DNA Cell Biol 12:1–51

    Article  PubMed  CAS  Google Scholar 

  10. Karplus PA, Daniels MJ, Herriot JR (1991) Science 251:60–66

    Article  PubMed  CAS  Google Scholar 

  11. Mueller EJ, Loida PJ, Sligar SG (1995) In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism and biochemistry. Plenum Press, New York, pp 83–124

    Chapter  Google Scholar 

  12. Jenkins CM, Waterman MR (1993) J Biol Chem 269:27401–27408

    Google Scholar 

  13. McIver L, Leadbeater C, Campopiano DJ, Baxter RL, Daff SN, Chapman SK, Munro AW (1998) Eur J Biochem 257:577–585

    Article  PubMed  CAS  Google Scholar 

  14. Lu P, Alterman MA, Chaurasia CS, Bambal RB, Hanzlik RP (1997) Arch Biochem Biophys 337:1–7

    Article  PubMed  CAS  Google Scholar 

  15. Nolan C (ed) (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  16. Zoller MJ, Smith M (1982) Nucleic Acids Res 10:6487–6500

    Article  PubMed  CAS  Google Scholar 

  17. Kunkel TA (1985) Proc Natl Acad Sci USA 82:488–492

    Article  PubMed  CAS  Google Scholar 

  18. Miles CS, Rouviere-Fourmy N, Lederer F, Matthews FS, Reid GA, Black MT, Chapman SK (1992) Biochem J 285:187–192

    PubMed  CAS  Google Scholar 

  19. Gibson TJ (1984) PhD thesis, University of Cambridge, UK

    Google Scholar 

  20. Kunkel TA, Roberts JD, Zakour RA (1987) Methods Enzymol 154:367–382

    Article  PubMed  CAS  Google Scholar 

  21. Vieira J, Messing J (1987) Methods Enzymol 153:3–11

    Article  PubMed  CAS  Google Scholar 

  22. Studier FW (1991) J Mol Biol 219:37–44

    Article  PubMed  CAS  Google Scholar 

  23. Studier FW, Moffatt BA (1986) J Mol Biol 189:113–130

    Article  PubMed  CAS  Google Scholar 

  24. Vieira J, Messing J (1982) Gene 19:259–268

    Article  PubMed  CAS  Google Scholar 

  25. Mead DA, Szczesnaskorupa E, Kemper B (1986) Protein Eng 1:67–74

    Article  PubMed  CAS  Google Scholar 

  26. Sanger F, Coulson AR, Barrell BG, Smith AJ, Roe BA (1980) J Mol Biol 143:161–178

    Article  PubMed  CAS  Google Scholar 

  27. Miles JS, Munro AW, Rospendowski BN, Smith WE, McKnight J, Thomson AJ (1992) Biochem J 288:503–509

    PubMed  CAS  Google Scholar 

  28. Smith GCM, Tew DG, Wolf CR (1994) Proc Natl Acad Sci USA 91:8710–8714

    Article  PubMed  CAS  Google Scholar 

  29. DeVoss JJ, Stok JE (1997) FASEB J 11:237

    Google Scholar 

  30. Narhi LO, Fulco AJ (1986) J Biol Chem 261:7160–7169

    PubMed  CAS  Google Scholar 

  31. Schwaneberg U, Schmidt-Dannert C, Scmitt J, Schmid RD (1999) Anal Biochem 269:359–366

    Article  PubMed  CAS  Google Scholar 

  32. Cupp-Vickery JR, Poulos TL (1995) Nat Struct Biol 2:144–152

    Article  PubMed  CAS  Google Scholar 

  33. He JS, Ruettinger RT, Liu HM, Fulco AJ (1989) Biochim Biophys Acta 1009:301–303

    Article  PubMed  CAS  Google Scholar 

  34. Yeom H, Sligar SG, Li H-Y, Poulos TL, Fulco AJ (1995) Biochemistry 34:14733–14740

    Article  PubMed  CAS  Google Scholar 

  35. Daff SN, Chapman SK, Turner KL, Holt RA, Govindaraj S, Poulos TL, Munro AW (1997) Biochemistry 36:13816–13823

    Article  PubMed  CAS  Google Scholar 

  36. Sligar SG (1976) Biochemistry 15:5399–5406

    Article  PubMed  CAS  Google Scholar 

  37. Tsai RL, Yu L-A, Gunsalus IC, Peisach J, Blumberg WE, Orme-Johnson WH, Beinert H (1970) Proc Natl Acad Sci USA 66:1157–1163

    Article  PubMed  CAS  Google Scholar 

  38. Lipscomb DJ (1980) Biochemistry 19:3590–3599

    Article  PubMed  CAS  Google Scholar 

  39. McKnight J, Cheesman MR, Thomson AJ, Miles JS, Munro AW (1993) Eur J Biochem 213:683–687

    Article  PubMed  CAS  Google Scholar 

  40. Cheesman MR, Greenwood C, Thomson AJ (1991) Adv Inorg Chem 36:201–255

    Article  CAS  Google Scholar 

  41. Vickery L, Salmon A, Sauer K (1975) Biochim Biophys Acta 386:87–98

    Article  PubMed  CAS  Google Scholar 

  42. Dawson JH, Sono M, Hager LP (1983) Inorg Chim Acta 79:184–186

    Article  Google Scholar 

  43. Sono M, Andersson LA, Dawson JH (1982) J Biol Chem 257:8308–8320

    PubMed  CAS  Google Scholar 

  44. Day P, Smith DW, Williams RJP (1967) Biochemistry 6:3747–3750

    Article  PubMed  CAS  Google Scholar 

  45. Gadsby PMA, Thomson AJ (1990) J Am Chem Soc 112:5003–5011

    Article  CAS  Google Scholar 

  46. Abe M, Kitagawa T, Kyogoku Y (1978) J Chem Phys 69:4526–4534

    Article  CAS  Google Scholar 

  47. Hudecek J, Baumruk V, Anzenbacher P, Munro AW (1999) Biochem Biophys Res Commun 243:811–815

    Article  Google Scholar 

  48. Atkins WM, Sligar SG (1988) J Biol Chem 263:18842–18849

    PubMed  CAS  Google Scholar 

  49. Noble MA, Turner KL, Chapman SK, Hanzlik RP, Munro AW (1998) Biochemistry 37:15799–15807

    Article  PubMed  CAS  Google Scholar 

  50. Munro AW, Lindsay JG (1996) Mol Microbiol 20:1115–1125

    Article  PubMed  CAS  Google Scholar 

  51. Okita RT, Clark JE, Rice-Okita J, Masters BSS (1991) Methods Enzymol 206:432–441

    Article  PubMed  CAS  Google Scholar 

  52. Matsunaga I, Ueda A, Fujiwara N, Sumimoto T, Ichihara K (1999) Lipids 34:841–846

    Article  PubMed  CAS  Google Scholar 

  53. Noble MA, Miles CS, Chapman SK, Lysek DA, Mackay AC, Reid GA, Hanzlik RP, Munro AW (1999) Biochem J 339:371–379

    Article  PubMed  CAS  Google Scholar 

  54. Kunst F, Ogasawara N, Moszer I, Albertini M, Alloni G, et al. (1997) Nature (London) 390:249–256

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pure & Applied Chemistry, University of Strathclyde, The Royal College, 204 George Street, Glasgow, G1 1XL, UK

    Iain D. G. Macdonald & Andrew W. Munro

  2. Department of Chemistry, Joseph Black Building, The University of Edinburgh, The King’s Buildings, West Mains Road, Edinburgh, EH9 3JJ, UK

    Amanda J. Green & Stephen K. Chapman

  3. Institute of Cell and Molecular Biology, The King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK

    Stuart L. Rivers & Graeme A. Reid

  4. Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich, UK

    Myles Cheesman

  5. Department of Chemistry, University of Minnesota, 400 Smith Hall, 207 Pleasant Street, Minneapolis, MN, 55455, USA

    Luca G. Quaroni

Authors
  1. Amanda J. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stuart L. Rivers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Myles Cheesman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Graeme A. Reid
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luca G. Quaroni
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Iain D. G. Macdonald
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephen K. Chapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrew W. Munro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew W. Munro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Green, A.J., Rivers, S.L., Cheesman, M. et al. Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis . JBIC 6, 523–533 (2001). https://doi.org/10.1007/s007750100229

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s007750100229

Keywords

Search

Navigation