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. 1997 Oct 15;327(Pt 2):537–544. doi: 10.1042/bj3270537

Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds.

C F Oliver 1, S Modi 1, W U Primrose 1, L Y Lian 1, G C Roberts 1
PMCID: PMC1218827  PMID: 9359427

Abstract

Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (omega-1, omega-2, omega-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47-->glutamate mutant at the omega-1, omega-2 and omega-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 A closer to the iron, than seen in the wild-type, although this difference is much smaller ( approximately 0.2 A) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.

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Selected References

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  1. Boddupalli S. S., Estabrook R. W., Peterson J. A. Fatty acid monooxygenation by cytochrome P-450BM-3. J Biol Chem. 1990 Mar 15;265(8):4233–4239. [PubMed] [Google Scholar]
  2. Capdevila J. H., Wei S., Helvig C., Falck J. R., Belosludtsev Y., Truan G., Graham-Lorence S. E., Peterson J. A. The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. J Biol Chem. 1996 Sep 13;271(37):22663–22671. doi: 10.1074/jbc.271.37.22663. [DOI] [PubMed] [Google Scholar]
  3. Cupp-Vickery J. R., Poulos T. L. Structure of cytochrome P450eryF involved in erythromycin biosynthesis. Nat Struct Biol. 1995 Feb;2(2):144–153. doi: 10.1038/nsb0295-144. [DOI] [PubMed] [Google Scholar]
  4. Dawson J. H. Probing structure-function relations in heme-containing oxygenases and peroxidases. Science. 1988 Apr 22;240(4851):433–439. doi: 10.1126/science.3358128. [DOI] [PubMed] [Google Scholar]
  5. Fulco A. J. P450BM-3 and other inducible bacterial P450 cytochromes: biochemistry and regulation. Annu Rev Pharmacol Toxicol. 1991;31:177–203. doi: 10.1146/annurev.pa.31.040191.001141. [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. Hasemann C. A., Kurumbail R. G., Boddupalli S. S., Peterson J. A., Deisenhofer J. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure. 1995 Jan 15;3(1):41–62. doi: 10.1016/s0969-2126(01)00134-4. [DOI] [PubMed] [Google Scholar]
  8. Ho P. P., Fulco A. J. Involvement of a single hydroxylase species in the hydroxylation of palmitate at the omega-1, omega-2 and omega-3 positions by a preparation from Bacillus megaterium. Biochim Biophys Acta. 1976 May 27;431(2):249–256. doi: 10.1016/0005-2760(76)90145-4. [DOI] [PubMed] [Google Scholar]
  9. Li H. Y., Darwish K., Poulos T. L. Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains. J Biol Chem. 1991 Jun 25;266(18):11909–11914. [PubMed] [Google Scholar]
  10. 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]
  11. Matson R. S., Hare R. S., Fulco A. J. Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium. Biochim Biophys Acta. 1977 Jun 22;487(3):487–494. doi: 10.1016/0005-2760(77)90218-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Miura Y., Fulco A. J. Omega-1, Omega-2 and Omega-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium. Biochim Biophys Acta. 1975 Jun 23;388(3):305–317. doi: 10.1016/0005-2760(75)90089-2. [DOI] [PubMed] [Google Scholar]
  14. Modi S., Behere D. V., Mitra S. Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies. Biochim Biophys Acta. 1989 Jul 6;996(3):214–225. doi: 10.1016/0167-4838(89)90250-1. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Narhi L. O., Wen L. P., Fulco A. J. Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene. Mol Cell Biochem. 1988 Jan;79(1):63–71. doi: 10.1007/BF00229399. [DOI] [PubMed] [Google Scholar]
  19. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem. 1964 Jul;239:2379–2385. [PubMed] [Google Scholar]
  20. 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]
  21. Poulos T. L., Finzel B. C., Howard A. J. High-resolution crystal structure of cytochrome P450cam. J Mol Biol. 1987 Jun 5;195(3):687–700. doi: 10.1016/0022-2836(87)90190-2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ruettinger R. T., Wen L. P., Fulco A. J. Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. J Biol Chem. 1989 Jul 5;264(19):10987–10995. [PubMed] [Google Scholar]
  24. Sariaslani F. S. Microbial cytochromes P-450 and xenobiotic metabolism. Adv Appl Microbiol. 1991;36:133–178. doi: 10.1016/s0065-2164(08)70453-2. [DOI] [PubMed] [Google Scholar]

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