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Volume 158, Issue 12

Molecular characterization of and , essential for C24-branched chain sterol-side-chain degradation in DSM 43269 Free

Abstract

A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of , which selectively degrades sterol side-chains. The and deletion mutants were each completely blocked in side-chain degradation of β-sitosterol and campesterol, but not of cholesterol. These results indicated a role for and in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in β-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of β-sitosterol was previously shown to proceed via aldolytic cleavage rather than by β-oxidation. Our results therefore suggest that and probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon–carbon bond cleavage of C24-branched chain sterols in strains, most likely acting as aldol-lyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.

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2012-12-01
2023-09-24
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References

  1. Ambrus G., Ilköy E., Jekkel A., Horváth G., Böcskei Z. ( 1995). Microbial transformation of β-sitosterol and stigmasterol into 26-oxygenated derivatives. Steroids 60:621–625 [View Article][PubMed]
     [Google Scholar]
  2. Arima K., Nagasawa M., Bae M., Tamura M. ( 1969). Microbial transformation of sterols. Part I. Decomposition of cholesterol by microorganisms. Agric Biol Chem 33:1636–1643 [View Article]
     [Google Scholar]
  3. Bekal S., Van Beeumen J., Samyn B., Garmyn D., Henini S., Diviès C., Prévost H. ( 1998). Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster. J Bacteriol 180:647–654[PubMed]
     [Google Scholar]
  4. Brooks C. J. W. ( 1979). Some aspects of mass spectrometry in research on steroids. Philos Trans R Soc Lond Ser A 293:53–67 [View Article]
     [Google Scholar]
  5. Capyk J. K., Kalscheuer R., Stewart G. R., Liu J., Kwon H., Zhao R., Okamoto S., Jacobs W. R. Jr, Eltis L. D., Mohn W. W. ( 2009). Mycobacterial cytochrome p450 125 (cyp125) catalyzes the terminal hydroxylation of c27 steroids. J Biol Chem 284:35534–35542 [View Article][PubMed]
     [Google Scholar]
  6. Chen C. S. ( 1985). The mechanism of degradation of side chains of phytosterols by microorganisms
     [Google Scholar]
  7. Dodson R. M., Muir R. D. ( 1958a). Microbiological transformations. II. The microbiological aromatization of steroids. J Am Chem Soc 80:5004–5005 [View Article]
     [Google Scholar]
  8. Dodson R. M., Muir R. D. ( 1958b). Microbiological transformations. III. The hydroxylation of steroids at C-9. J Am Chem Soc 80:6148 [View Article]
     [Google Scholar]
  9. Finn R. D., Tate J., Mistry J., Coggill P. C., Sammut S. J., Hotz H. R., Ceric G., Forslund K., Eddy S. R. & other authors ( 2008). The Pfam protein families database. Nucleic Acids Res 36:Database issueD281–D288 [View Article][PubMed]
     [Google Scholar]
  10. Fujimoto Y., Chen C.-S., Szeleczky Z., DiTullio D., Sih C. J. ( 1982a). Microbial degradation of the phytosterol side chain. 1. Enzymatic conversion of 3-oxo-24-ethylcholest-4-en-26-oic acid into 3-oxochol-4-en-24-oic acid and androst-4-ene-3,17-dione. J Am Chem Soc 104:4718–4720 [View Article]
     [Google Scholar]
  11. Fujimoto Y., Chen C.-S., Gopalan A. S., Sih C. J. ( 1982b). Microbial degradation of the phytosterol side chain. II. Incorporation of [14C]-NaHCO3 onto the C-28 position. J Am Chem Soc 104:4720–4722 [CrossRef]
     [Google Scholar]
  12. Haapalainen A. M., Meriläinen G., Wierenga R. K. ( 2006). The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends Biochem Sci 31:64–71[PubMed] [CrossRef]
     [Google Scholar]
  13. Hacking A. J., Quayle J. R. ( 1974). Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1. Biochem J 139:399–405[PubMed]
     [Google Scholar]
  14. Johnston J. B., Ouellet H., Ortiz de Montellano P. R. ( 2010). Functional redundancy of steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by biochemical and genetic analyses. J Biol Chem 285:36352–36360 [View Article][PubMed]
     [Google Scholar]
  15. Martin C. K. A. ( 1977). Microbial cleavage of sterol side chains. Adv Appl Microbiol 22:29–58 [View Article][PubMed]
     [Google Scholar]
  16. Martin C. K. A., Wagner F. ( 1976). Microbial transformation of β-sitosterol by Nocardia sp. M29. Eur J Appl Microbiol 2:243–255 [View Article]
     [Google Scholar]
  17. Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., Yano K. ( 1995). Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. Appl Environ Microbiol 61:2079–2085[PubMed]
     [Google Scholar]
  18. McLean K. J., Lafite P., Levy C., Cheesman M. R., Mast N., Pikuleva I. A., Leys D., Munro A. W. ( 2009). The structure of Mycobacterium tuberculosis CYP125: molecular basis for cholesterol binding in a P450 needed for host infection. J Biol Chem 284:35524–35533 [View Article][PubMed]
     [Google Scholar]
  19. McLeod M. P., Warren R. L., Hsiao W. W., Araki N., Myhre M., Fernandes C., Miyazawa D., Wong W., Lillquist A. L. & other authors ( 2006). The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A 103:15582–15587 [View Article][PubMed]
     [Google Scholar]
  20. Nesbitt N. M., Yang X., Fontán P., Kolesnikova I., Smith I., Sampson N. S., Dubnau E. ( 2010). A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol. Infect Immun 78:275–282 [View Article][PubMed]
     [Google Scholar]
  21. Ouellet H., Guan S., Johnston J. B., Chow E. D., Kells P. M., Burlingame A. L., Cox J. S., Podust L. M., de Montellano P. R. ( 2010). Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one. Mol Microbiol 77:730–742 [View Article][PubMed]
     [Google Scholar]
  22. Peretó J., López-García P., Moreira D. ( 2005). Phylogenetic analysis of eukaryotic thiolases suggests multiple proteobacterial origins. J Mol Evol 61:65–74[PubMed] [CrossRef]
     [Google Scholar]
  23. Petrusma M., Dijkhuizen L., van der Geize R. ( 2009). Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9α-hydroxylase, a two-component iron–sulfur-containing monooxygenase with subtle steroid substrate specificity. Appl Environ Microbiol 75:5300–5307[PubMed] [CrossRef]
     [Google Scholar]
  24. Rosłoniec K. Z., Wilbrink M. H., Capyk J. K., Mohn W. W., Ostendorf M., van der Geize R., Dijkhuizen L., Eltis L. D. ( 2009). Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1. Mol Microbiol 74:1031–1043 [View Article][PubMed]
     [Google Scholar]
  25. 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]
  26. Seedorf U., Brysch P., Engel T., Schrage K., Assmann G. ( 1994). Sterol carrier protein X is peroxisomal 3-oxoacyl coenzyme A thiolase with intrinsic sterol carrier and lipid transfer activity. J Biol Chem 269:21277–21283[PubMed]
     [Google Scholar]
  27. Sih C. J., Tai H. H., Tsong Y. Y. ( 1967). The mechanism of microbial conversion of cholesterol into 17-keto steroids. J Am Chem Soc 89:1957–1958[PubMed] [CrossRef]
     [Google Scholar]
  28. Sih C. J., Tai H. H., Tsong Y. Y., Lee S. S., Coombe R. G. ( 1968a). Mechanisms of steroid oxidation by microorganisms. XIV. Pathway of cholesterol side-chain degradation. Biochemistry 7:808–818 [View Article][PubMed]
     [Google Scholar]
  29. Sih C. J., Wang K. C., Tai H. H. ( 1968b). Mechanisms of steroid oxidation by microorganisms. XIII. C22 acid intermediates in the degradation of the cholesterol side chain. Biochemistry 7:796–807 [View Article][PubMed]
     [Google Scholar]
  30. Song J., Wadhwa L., Bejjani B. A., O’Brien W. E. ( 2003). Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization. J Chromatogr B Analyt Technol Biomed Life Sci 791:127–135 [View Article][PubMed]
     [Google Scholar]
  31. Stegink L. D., Coon M. J. ( 1968). Stereospecificity and other properties of highly purified β-hydroxy-β-methylglutaryl coenzyme A cleavage enzyme from bovine liver. J Biol Chem 243:5272–5279[PubMed]
     [Google Scholar]
  32. Tamura K., Dudley J., Nei M., Kumar S. ( 2007). mega4: molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol 24:1596–1599 [View Article][PubMed]
     [Google Scholar]
  33. Thompson J. D., Higgins D. G., Gibson T. J. ( 1994). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [View Article][PubMed]
     [Google Scholar]
  34. Uhía I., Galán B., Kendall S. L., Stoker N. G., García J. L. ( 2012). Cholesterol metabolism in Mycobacterium smegmatis . Environ Microbiol Rep 4:168–182 [View Article]
     [Google Scholar]
  35. van der Geize R., Dijkhuizen L. ( 2004). Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Curr Opin Microbiol 7:255–261 [View Article][PubMed]
     [Google Scholar]
  36. van der Geize R., Hessels G. I., van Gerwen R., van der Meijden P., Dijkhuizen L. ( 2001). Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker. FEMS Microbiol Lett 205:197–202[PubMed] [CrossRef]
     [Google Scholar]
  37. van der Geize R., Yam K., Heuser T., Wilbrink M. H., Hara H., Anderton M. C., Sim E., Dijkhuizen L., Davies J. E. & other authors ( 2007). A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci U S A 104:1947–1952 [View Article][PubMed]
     [Google Scholar]
  38. van der Geize R., de Jong W., Hessels G. I., Grommen A. W., Jacobs A. A., Dijkhuizen L. ( 2008). A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality. Nucleic Acids Res 36:e151[PubMed] [CrossRef]
     [Google Scholar]
  39. Wanders R. J., Denis S., Wouters F., Wirtz K. W., Seedorf U. ( 1997). Sterol carrier protein X (SCPx) is a peroxisomal branched-chain β-ketothiolase specifically reacting with 3-oxo-pristanoyl-CoA: a new, unique role for SCPx in branched-chain fatty acid metabolism in peroxisomes. Biochem Biophys Res Commun 236:565–569[PubMed] [CrossRef]
     [Google Scholar]
  40. Wilbrink M. H., Petrusma M., Dijkhuizen L., van der Geize R. ( 2011). FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains. Appl Environ Microbiol 77:4455–4464[PubMed] [CrossRef]
     [Google Scholar]
  41. Zaretskaya I. I., Kogan L. M., Tkhomirova O. B., Sis J. D., Wulfson N. S., Zaretskii V. I., Zaikin V. G., Skryabin G. K., Torgov I. V. ( 1967). Microbial hydroxylation of the cholesterol side chain. Tetrahedon 24:1595–1600[PubMed] [CrossRef]
     [Google Scholar]
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Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269
Microbiology 158, 3054 (2012); https://doi.org/10.1099/mic.0.059501-0
/content/journal/micro/10.1099/mic.0.059501-0
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