1887

Abstract

Cloning and sequencing of the operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by sp. strain HE5. The cytochrome P450 (P450) and the FeS ferredoxin (Fd), encoded by and , respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450 system. Analysis of the operon has now revealed the gene , encoding the ferredoxin reductase of this morpholine monooxygenase. The genes , and were identical to the corresponding genes from sp. strain RP1. Almost identical genes in PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in sp. strain HE5. Analysis of the downstream sequences of revealed differences in this gene region between sp. strain HE5 and sp. strain RP1 on the one hand, and on the other hand, indicating insertions or deletions after recombination. Downstream of the genes, the gene ′, encoding a putative glutamine synthetase, was identified in all studied strains. The gene of sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The values of FdR for the substrate NADH (37·7±4·1 μM) and the artificial electron acceptors potassium ferricyanide (14·2±1·1 μM) and cytochrome (28·0±3·6 μM) were measured. FdR was shown to interact functionally with its natural redox partner, the FeS protein Fd, and with the FeS protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd in activity assays. These results indicated that FdR can utilize different ferredoxins, but that Fd requires the specific NADH : ferredoxin oxidoreductase FdR from the P450 system for efficient catalytic function.

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2005-08-01
2020-04-02
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References

  1. Aliverti A., Curti B., Vanoni M. A. 1999; Identifying and quantitating FAD and FMN in simple and iron-sulfur-containing flavoproteins. In Methods in Molecular Biology pp9–23 Edited by Chapman S. K., Reid G. A.. Totowa, NJ: Humana Press Inc;
    [Google Scholar]
  2. Apajalahti J. H., Salkinoja-Salonen M. S. 1987; Dechlorination and para-hydroxylation of polychlorinated phenols by Rhodococcus chlorophenolicus. J Bacteriol169:675–681
    [Google Scholar]
  3. Cech J. S., Hartman P., Slosarek M., Chudoba J. 1988; Isolation and identification of a morpholine-degrading bacterium. Appl Environ Microbiol54:619–621
    [Google Scholar]
  4. Cole S. T., Brosch R., Parkhill J. 22 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature393:537–544[CrossRef]
    [Google Scholar]
  5. Combourieu B., Poupin P., Besse P., Sancelme M., Veschambre H., Truffaut N., Delort A. M. 1998a; Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR. Biodegradation 9433–442[CrossRef]
    [Google Scholar]
  6. Combourieu B., Besse P., Sancelme M., Veschambre H., Delort A. M., Poupin P., Truffaut N. 1998b; Morpholine degradation pathway of Mycobacterium aurum MO1: direct evidence of intermediates by in situ 1H nuclear magnetic resonance. Appl Environ Microbiol64:153–158
    [Google Scholar]
  7. Combourieu B., Besse P., Sancelme M., Godin J. P., Monteil A., Veschambre H., Delort A. M. 2000; Common degradative pathways of morpholine, thiomorpholine, and piperidine by Mycobacterium aurum MO1: evidence from 1H-nuclear magnetic resonance and ionspray mass spectrometry performed directly on the incubation medium. Appl Environ Microbiol66:3187–3193[CrossRef]
    [Google Scholar]
  8. Debbab M. 2003; Weitere Untersuchungen zum Stoffwechsel von Mycobacterium sp Stamm HE5 Diploma thesis Martin-Luther-Universität Halle-Wittenberg;
    [Google Scholar]
  9. Dym O., Eisenberg D. 2001; Sequence-structure analysis of FAD-containing proteins. Protein Sci10:1712–1728[CrossRef]
    [Google Scholar]
  10. Fischer F., Raimondi D., Aliverti A., Zanetti G. 2002; Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. Eur J Biochem269:3005–3013[CrossRef]
    [Google Scholar]
  11. Geren L., Tuls J., O'Brien P., Millet F., Peterson J. A. 1986; The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase. J Biol Chem261:15491–15495
    [Google Scholar]
  12. Grinberg A. V., Hannemann F., Schiffler B., Müller J., Heinemann U., Bernhardt R. 2000; Adrenodoxin: structure, stability, and electron transfer properties. Proteins40:590–612[CrossRef]
    [Google Scholar]
  13. Häggblom M. M., Nohynek L. J., Palleroni N. J., Kronqvist K., Nurmiaho-Lassila E. L., Salkinoja-Salonen M. S., Klatte S., Kroppenstedt R. M. 1994; Transfer of polychlorophenol-degrading Rhodococcus chlorophenolicus. Apajalahti et al., 1986) to the genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov. Int J Syst Bacteriol44:485–493[CrossRef]
    [Google Scholar]
  14. Hawkes D. B., Adams G. W., Burlingame A. L., Ortiz de Montellano P. R., De Voss J. J. 2002; Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization. J Biol Chem277:27725–27732[CrossRef]
    [Google Scholar]
  15. Hughes V. M., Stevenson K., Sharp J. M. 2001; Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria. J Microbiol Methods44:209–215[CrossRef]
    [Google Scholar]
  16. Ikeda H., Ishikawa J., Hanamoto A., Shinose M., Kikuchi H., Shiba T., Sakaki Y., Hattori M., Omura S. 2003; Complete genome sequence and comparative analysis of the industrial microorganism. Streptomyces avermitilis. Nat Biotechnol21:526–531[CrossRef]
    [Google Scholar]
  17. Jackson C. J., Lamb D. C., Marczylo T. H., Parker J. E., Manning N. L., Kelly D. E., Kelly S. L. 2003; Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from. Mycobacterium smegmatis. Biochem Biophys Res Commun301:558–563[CrossRef]
    [Google Scholar]
  18. Knapp J. S., Brown V. R. 1988; Morpholine biodegradation. Int Biodeterior24:299–306[CrossRef]
    [Google Scholar]
  19. Koga H., Rauchfuss B., Gunsalus I. C. 1985; P450cam gene cloning and expression in. Pseudomonas putida and Escherichia coli. Biochem Biophys Res Commun130:412–417[CrossRef]
    [Google Scholar]
  20. Lamb D. C., Ikeda H., Nelson D. R., Ishikawa J., Skaug T., Jackson C., Omura S., Waterman M. R., Kelly S. L. 2003; Cytochrome P450 complement (CYPome) of the avermectin-producer. Streptomyces avermitilis and comparison to that of Streptomyces coelicolor. A3(2). Biochem Biophys Res Commun307:610–619[CrossRef]
    [Google Scholar]
  21. Larsen M. H. 2000; Some common methods in mycobacterial genetics. In Molecular Genetics of Mycobacteria pp313–317 Edited by Hatfull G. F. J., Jacobs W. R. Jr. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  22. Lei L., Waterman M. R., Fulco A. J., Kelly S. L., Lamb D. C. 2004; Availability of specific reductases controls the temporal activity of the cytochrome P450 complement of. Streptomyces coelicolor. A3(2). Proc Natl Acad Sci U S A101:494–499[CrossRef]
    [Google Scholar]
  23. McLean K. J., Scrutton N. S., Munro A. W. 2003; Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. Biochem J372:317–327[CrossRef]
    [Google Scholar]
  24. Munro A. W., Lindsay J. G. 1996; Bacterial cytochromes P-450. Mol Microbiol20:1115–1125[CrossRef]
    [Google Scholar]
  25. Nagy I., Schoofs G., Compernolle F., Proost P., Vanderleyden J, de Mot R. 1995; 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 Bacteriol177:676–687
    [Google Scholar]
  26. O'Keefe D. P., Harder P. A. 1991; Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes. Mol Microbiol5:2099–2105[CrossRef]
    [Google Scholar]
  27. Omura S., Ikeda H., Ishikawa J. 11 other authors 2001; Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A98:12215–12220[CrossRef]
    [Google Scholar]
  28. Peterson J. A., Lu J. Y., Geisselsoder J., Graham-Lorence S., Carmona C., Witney F., Lorence M. C. 1992; Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J Biol Chem267:14193–14203
    [Google Scholar]
  29. Poelarends G. J., Zandstra M., Bosma T., Kulakov L. A., Larkin M. J., Marchesi J. R., Weightman A. J., Jannsen D. B. 2000; Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism. J Bacteriol182:2725–2731[CrossRef]
    [Google Scholar]
  30. Poupin P., Truffaut N., Combourieu B., Besse P., Sancelme M., Veschambre H., Delort A. M. 1998; Degradation of morpholine by an environmental Mycobacterium strain involves a cytochrome P-450. Appl Environ Microbiol64:159–165
    [Google Scholar]
  31. Poupin P., Godon J. J., Zumstein E., Truffaut N. 1999a; Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450. Can J Microbiol45:209–216[CrossRef]
    [Google Scholar]
  32. Poupin P., Ducrocq V., Hallier-Soulier S., Truffaut N. 1999b; Cloning and characterization of the genes encoding a cytochrome P450 (PipA) involved in piperidine and pyrrolidine utilization and its regulatory protein (PipR) in. Mycobacterium smegmatis mc2155. J Bacteriol181:3419–3426
    [Google Scholar]
  33. Ramachandra M., Seetharam R., Emptage M. H., Sariaslani F. S. 1991; Purification and characterization of a soybean flour-inducible ferredoxin reductase of. Streptomyces griseus. J Bacteriol173:7106–7112
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Schiffler B., Bernhardt R. 2003; Bacterial (CYP101) and mitochondrial P450 systems – how comparable are they?. Biochem Biophys Res Commun312:223–228[CrossRef]
    [Google Scholar]
  36. Schräder, T., Schuffenhauer G., Sielaff B., Andreesen J. R. 2000; High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated. Mycobacterium. sp. strain HE5. Microbiology146:1091–1098
    [Google Scholar]
  37. Schuffenhauer G., Schräder T., Andreesen J. R. 1999; Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5. Arch Microbiol171:417–423[CrossRef]
    [Google Scholar]
  38. Sevrioukova I. F., Li H., Poulos T. L. 2004; Crystal structure of putidaredoxin reductase from Pseudomonas putida, the final structural component of the cytochrome P450cam monooxygenase. J Mol Biol336:889–902[CrossRef]
    [Google Scholar]
  39. Sielaff B., Andreesen J. R. 2005; Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from. Mycobacterium sp. strain HE5. FEBS J272:1148–1159[CrossRef]
    [Google Scholar]
  40. Sielaff B., Andreesen J. R., Schräder T. 2001; A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine. Appl Microbiol Biotechnol56:458–464[CrossRef]
    [Google Scholar]
  41. Trigui M., Pulvin S., Truffaut N., Thomas D., Poupin P. 2004; Molecular cloning, nucleotide sequencing and expression of genes encoding a cytochrome P450 system involved in secondary amine utilization in. Mycobacterium sp. strain RP1. Res Microbiol155:1–9[CrossRef]
    [Google Scholar]
  42. Uhlmann H., Kraft R., Bernhardt R. 1994; C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450. J Biol Chem269:22557–22564
    [Google Scholar]
  43. Urlacher V. B., Lutz-Wahl S., Schmid R. D. 2004; Microbial P450 enzymes in biotechnology. Appl Microbiol Biotechnol64:317–325[CrossRef]
    [Google Scholar]
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