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. 1999 Apr 15;339(Pt 2):371–379.

Roles of key active-site residues in flavocytochrome P450 BM3.

M A Noble 1, C S Miles 1, S K Chapman 1, D A Lysek 1, A C MacKay 1, G A Reid 1, R P Hanzlik 1, A W Munro 1
PMCID: PMC1220167  PMID: 10191269

Abstract

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

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Selected References

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  1. Barstow D. A., Clarke A. R., Chia W. N., Wigley D., Sharman A. F., Holbrook J. J., Atkinson T., Minton N. P. Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene. Gene. 1986;46(1):47–55. doi: 10.1016/0378-1119(86)90165-4. [DOI] [PubMed] [Google Scholar]
  2. Black M. T., Gunn F. J., Chapman S. K., Reid G. A. Structural basis for the kinetic differences between flavocytochromes b2 from the yeasts Hansenula anomala and Saccharomyces cerevisiae. Biochem J. 1989 Nov 1;263(3):973–976. doi: 10.1042/bj2630973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Capdevila J. H., Falck J. R., Estabrook R. W. Cytochrome P450 and the arachidonate cascade. FASEB J. 1992 Jan 6;6(2):731–736. doi: 10.1096/fasebj.6.2.1537463. [DOI] [PubMed] [Google Scholar]
  4. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S., Barry C. E., 3rd Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998 Jun 11;393(6685):537–544. doi: 10.1038/31159. [DOI] [PubMed] [Google Scholar]
  5. Daff S. N., Chapman S. K., Turner K. L., Holt R. A., Govindaraj S., Poulos T. L., Munro A. W. Redox control of the catalytic cycle of flavocytochrome P-450 BM3. Biochemistry. 1997 Nov 11;36(45):13816–13823. doi: 10.1021/bi971085s. [DOI] [PubMed] [Google Scholar]
  6. Graham-Lorence S., Truan G., Peterson J. A., Falck J. R., Wei S., Helvig C., Capdevila J. H. An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase. J Biol Chem. 1997 Jan 10;272(2):1127–1135. doi: 10.1074/jbc.272.2.1127. [DOI] [PubMed] [Google Scholar]
  7. Guengerich F. P., Shimada T. Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol. 1991 Jul-Aug;4(4):391–407. doi: 10.1021/tx00022a001. [DOI] [PubMed] [Google Scholar]
  8. Hart K. W., Clarke A. R., Wigley D. B., Waldman A. D., Chia W. N., Barstow D. A., Atkinson T., Jones J. B., Holbrook J. J. A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase. Biochim Biophys Acta. 1987 Aug 21;914(3):294–298. doi: 10.1016/0167-4838(87)90289-5. [DOI] [PubMed] [Google Scholar]
  9. Imai M., Shimada H., Watanabe Y., Matsushima-Hibiya Y., Makino R., Koga H., Horiuchi T., Ishimura Y. Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7823–7827. doi: 10.1073/pnas.86.20.7823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Karlson U., Dwyer D. F., Hooper S. W., Moore E. R., Timmis K. N., Eltis L. D. Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate. J Bacteriol. 1993 Mar;175(5):1467–1474. doi: 10.1128/jb.175.5.1467-1474.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Li H., Poulos T. L. The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat Struct Biol. 1997 Feb;4(2):140–146. doi: 10.1038/nsb0297-140. [DOI] [PubMed] [Google Scholar]
  13. Lu P., Alterman M. A., Chaurasia C. S., Bambal R. B., Hanzlik R. P. Heme-coordinating analogs of lauric acid as inhibitors of fatty acid omega-hydroxylation. Arch Biochem Biophys. 1997 Jan 1;337(1):1–7. doi: 10.1006/abbi.1996.9768. [DOI] [PubMed] [Google Scholar]
  14. Macheroux P., Massey V., Thiele D. J., Volokita M. Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization. Biochemistry. 1991 May 7;30(18):4612–4619. doi: 10.1021/bi00232a036. [DOI] [PubMed] [Google Scholar]
  15. Marletta M. A. Nitric oxide synthase structure and mechanism. J Biol Chem. 1993 Jun 15;268(17):12231–12234. [PubMed] [Google Scholar]
  16. Maves S. A., Yeom H., McLean M. A., Sligar S. G. Decreased substrate affinity upon alteration of the substrate-docking region in cytochrome P450(BM-3). FEBS Lett. 1997 Sep 8;414(2):213–218. doi: 10.1016/s0014-5793(97)00999-x. [DOI] [PubMed] [Google Scholar]
  17. Miles C. S., Rouvière-Fourmy N., Lederer F., Mathews F. S., Reid G. A., Black M. T., Chapman S. K. Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2. Biochem J. 1992 Jul 1;285(Pt 1):187–192. doi: 10.1042/bj2850187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miles J. S., Munro A. W., Rospendowski B. N., Smith W. E., McKnight J., Thomson A. J. Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization. Biochem J. 1992 Dec 1;288(Pt 2):503–509. doi: 10.1042/bj2880503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Modi S., Primrose W. U., Boyle J. M., Gibson C. F., Lian L. Y., Roberts G. C. NMR studies of substrate binding to cytochrome P450 BM3: comparisons to cytochrome P450 cam. Biochemistry. 1995 Jul 18;34(28):8982–8988. doi: 10.1021/bi00028a006. [DOI] [PubMed] [Google Scholar]
  20. Modi S., Sutcliffe M. J., Primrose W. U., Lian L. Y., Roberts G. C. The catalytic mechanism of cytochrome P450 BM3 involves a 6 A movement of the bound substrate on reduction. Nat Struct Biol. 1996 May;3(5):414–417. doi: 10.1038/nsb0596-414. [DOI] [PubMed] [Google Scholar]
  21. Munro A. W., Daff S., Coggins J. R., Lindsay J. G., Chapman S. K. Probing electron transfer in flavocytochrome P-450 BM3 and its component domains. Eur J Biochem. 1996 Jul 15;239(2):403–409. doi: 10.1111/j.1432-1033.1996.0403u.x. [DOI] [PubMed] [Google Scholar]
  22. Munro A. W., Lindsay J. G. Bacterial cytochromes P-450. Mol Microbiol. 1996 Jun;20(6):1115–1125. doi: 10.1111/j.1365-2958.1996.tb02632.x. [DOI] [PubMed] [Google Scholar]
  23. Munro A. W., Lindsay J. G., Coggins J. R., Kelly S. M., Price N. C. Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3. Biochim Biophys Acta. 1996 Sep 5;1296(2):127–137. doi: 10.1016/0167-4838(96)00061-1. [DOI] [PubMed] [Google Scholar]
  24. Munro A. W., Lindsay J. G., Coggins J. R., Kelly S. M., Price N. C. Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3. FEBS Lett. 1994 Apr 18;343(1):70–74. doi: 10.1016/0014-5793(94)80609-8. [DOI] [PubMed] [Google Scholar]
  25. Munro A. W., Malarkey K., McKnight J., Thomson A. J., Kelly S. M., Price N. C., Lindsay J. G., Coggins J. R., Miles J. S. The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the 'covalent switching' hypothesis of P-450 electron transfer. Biochem J. 1994 Oct 15;303(Pt 2):423–428. doi: 10.1042/bj3030423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nagy I., Schoofs G., Compernolle F., Proost P., Vanderleyden J., de Mot R. Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase. J Bacteriol. 1995 Feb;177(3):676–687. doi: 10.1128/jb.177.3.676-687.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Narhi L. O., Fulco A. J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem. 1986 Jun 5;261(16):7160–7169. [PubMed] [Google Scholar]
  28. Nelson D. R., Kamataki T., Waxman D. J., Guengerich F. P., Estabrook R. W., Feyereisen R., Gonzalez F. J., Coon M. J., Gunsalus I. C., Gotoh O. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 1993 Jan-Feb;12(1):1–51. doi: 10.1089/dna.1993.12.1. [DOI] [PubMed] [Google Scholar]
  29. Noble M. A., Quaroni L., Chumanov G. D., Turner K. L., Chapman S. K., Hanzlik R. P., Munro A. W. Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3. Biochemistry. 1998 Nov 10;37(45):15799–15807. doi: 10.1021/bi980462d. [DOI] [PubMed] [Google Scholar]
  30. Okita R. T., Clark J. E., Okita J. R., Masters B. S. Omega- and (omega-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography. Methods Enzymol. 1991;206:432–441. doi: 10.1016/0076-6879(91)06112-g. [DOI] [PubMed] [Google Scholar]
  31. Oliver C. F., Modi S., Primrose W. U., Lian L. Y., Roberts G. C. Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem J. 1997 Oct 15;327(Pt 2):537–544. doi: 10.1042/bj3270537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oliver C. F., Modi S., Sutcliffe M. J., Primrose W. U., Lian L. Y., Roberts G. C. A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation. Biochemistry. 1997 Feb 18;36(7):1567–1572. doi: 10.1021/bi962826c. [DOI] [PubMed] [Google Scholar]
  33. Park S. Y., Shimizu H., Adachi S., Nakagawa A., Tanaka I., Nakahara K., Shoun H., Obayashi E., Nakamura H., Iizuka T. Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum. Nat Struct Biol. 1997 Oct;4(10):827–832. doi: 10.1038/nsb1097-827. [DOI] [PubMed] [Google Scholar]
  34. Ravichandran K. G., Boddupalli S. S., Hasermann C. A., Peterson J. A., Deisenhofer J. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. Science. 1993 Aug 6;261(5122):731–736. doi: 10.1126/science.8342039. [DOI] [PubMed] [Google Scholar]
  35. Sevrioukova I., Shaffer C., Ballou D. P., Peterson J. A. Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. Biochemistry. 1996 Jun 4;35(22):7058–7068. doi: 10.1021/bi960060a. [DOI] [PubMed] [Google Scholar]
  36. Smékal O., Yasin M., Fewson C. A., Reid G. A., Chapman S. K. L-mandelate dehydrogenase from Rhodotorula graminis: comparisons with the L-lactate dehydrogenase (flavocytochrome b2) from Saccharomyces cerevisiae. Biochem J. 1993 Feb 15;290(Pt 1):103–107. doi: 10.1042/bj2900103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stenberg K., Clausen T., Lindqvist Y., Macheroux P. Involvement of Tyr24 and Trp108 in substrate binding and substrate specificity of glycolate oxidase. Eur J Biochem. 1995 Mar 1;228(2):408–416. [PubMed] [Google Scholar]
  38. Tew D. G. Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications. Biochemistry. 1993 Sep 28;32(38):10209–10215. doi: 10.1021/bi00089a042. [DOI] [PubMed] [Google Scholar]
  39. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  40. Watanabe I., Nara F., Serizawa N. Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene. 1995 Sep 22;163(1):81–85. doi: 10.1016/0378-1119(95)00394-l. [DOI] [PubMed] [Google Scholar]
  41. Xia Z. X., Mathews F. S. Molecular structure of flavocytochrome b2 at 2.4 A resolution. J Mol Biol. 1990 Apr 20;212(4):837–863. doi: 10.1016/0022-2836(90)90240-M. [DOI] [PubMed] [Google Scholar]
  42. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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