1887

Abstract

The lipid-rich cell wall of mycobacteria is essential not only for virulence but also for survival. Whilst anabolic pathways for mycobacterial lipid biosynthesis have been well studied, there has been little research looking into lipid catabolism. The genome of encodes multiple enzymes with putative roles in the -oxidation of fatty acids. In this report we explore the functionality of FadB2, one of five homologues of a -hydroxybutyryl-CoA dehydrogenase, an enzyme that catalyses the third step in the -oxidation cycle. Purified FadB2 catalysed the NAD-dependent dehydration of -hydroxybutyryl-CoA to acetoacetyl-CoA at pH 10. Mutation of the active-site serine-122 residue resulted in loss of enzyme activity, consistent with the function of FadB2 as a fatty acyl dehydrogenase involved in the -oxidation of fatty acids. Surprisingly, purified FadB2 also catalysed the reverse reaction, converting acetoacetyl-CoA to -hydroxybutyryl-CoA, albeit in a lower pH range of 5.5–6.5. Additionally, a null mutant of was generated in . However, the mutant showed no significant differences from the wild-type strain with regard to lipid composition, utilization of different fatty acid carbon sources and tolerance to various stresses; the absence of any phenotype in the mutant strain could be due to the potential redundancy between the five paralogues.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.038802-0
2010-07-01
2020-09-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/7/1975.html?itemId=/content/journal/micro/10.1099/mic.0.038802-0&mimeType=html&fmt=ahah

References

  1. Bardarov S., Bardarov S. Jr, Pavelka M. S. Jr, Sambandamurthy V., Larsen M., Tufariello J., Chan J., Hatfull G., Jacobs W. R. Jr. 2002; Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M.bovis BCG and M. smegmatis. Microbiology148:3007–3017
    [Google Scholar]
  2. Barycki J. J., O'Brien L. K., Bratt J. M., Zhang R., Sanishvili R., Strauss A. W., Banaszak L. J.. 1999; Biochemical characterization and crystal structure determination of human heart short chain l-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism. Biochemistry38:5786–5798
    [Google Scholar]
  3. Bhatt A., Molle V., Besra G. S., Jacobs W. R. Jr, Kremer L.. 2007; The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. Mol Microbiol64:1442–1454
    [Google Scholar]
  4. Bishai W.. 2000; Lipid lunch for persistent pathogen. Nature406:683–685
    [Google Scholar]
  5. Black P. N., DiRusso C. C.. 1994; Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochim Biophys Acta1210:123–145
    [Google Scholar]
  6. Bloch H., Segal W.. 1956; Biochemical differentiation of Mycobacterium tuberculosis grown in vivo and in vitro. J Bacteriol72:132–141
    [Google Scholar]
  7. Chang J. C., Miner M. D., Pandey A. K., Gill W. P., Harik N. S., Sassetti C. M., Sherman D. R.. 2009; igr genes and Mycobacterium tuberculosis cholesterol metabolism. J Bacteriol191:5232–5239
    [Google Scholar]
  8. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S.. other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature393:537–544
    [Google Scholar]
  9. DiRusso C. C.. 1990; Primary sequence of the Escherichia coli fadBA operon, encoding the fatty acid-oxidizing multienzyme complex, indicates a high degree of homology to eucaryotic enzymes. J Bacteriol172:6459–6468
    [Google Scholar]
  10. El-Fakhri M., Middleton B.. 1982; The existence of an inner-membrane-bound, long acyl-chain-specific 3-hydroxyacyl-CoA dehydrogenase in mammalian mitochondria. Biochim Biophys Acta713:270–279
    [Google Scholar]
  11. Fisher M. A., Plikaytis B. B., Shinnick T. M.. 2002; Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. J Bacteriol184:4025–4032
    [Google Scholar]
  12. Garnier J., Gibrat J. F., Robson B.. 1996; GOR secondary structure prediction method version IV. Methods Enzymol266:540–553
    [Google Scholar]
  13. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., Bairoch A.. 2005; Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook pp571–607 Edited by Walker J. M.. Totowa, NJ: Humana Press;
  14. Gouet P., Courcelle E., Stuart D. I., Metoz F.. 1999; ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics15:305–308
    [Google Scholar]
  15. Heath R. J., Rock C. O.. 2004; Fatty acid biosynthesis as a target for novel antibacterials. Curr Opin Investig Drugs5:146–153
    [Google Scholar]
  16. Labarga A., Valentin F., Anderson M., Lopez R.. 2007; Web services at the European bioinformatics institute. Nucleic Acids Res35:W6–W11
    [Google Scholar]
  17. Liu X., Chu X., Yu W., Li P., Li D.. 2004; Expression and purification of His-tagged rat mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase wild-type and Ser137 mutant proteins. Protein Expr Purif37:344–351
    [Google Scholar]
  18. Luthra A., Malik S. S., Ramachandran R.. 2008; Cloning, purification and comparative structural analysis of two hypothetical proteins from Mycobacterium tuberculosis found in the human granuloma during persistence and highly up-regulated under carbon-starvation conditions. Protein Expr Purif62:64–74
    [Google Scholar]
  19. Manganelli R., Voskuil M. I., Schoolnik G. K., Smith I.. 2001; The Mycobacterium tuberculosis ECF sigma factor σE: role in global gene expression and survival in macrophages. Mol Microbiol41:423–437
    [Google Scholar]
  20. McGuire B. S., Carroll J. E., Chancey V. F., Howard J. C.. 1990; Mitochondrial enzymes responsible for oxidizing medium-chain fatty acids in developing rat skeletal muscle, heart, and liver. J Nutr Biochem1:410–414
    [Google Scholar]
  21. McKinney J. D., Honer zu Bentrup K., Munoz-Elias E. J., Miczak A., Chen B., Chan W. T., Swenson D., Sacchettini J. C., Jacobs W. R. Jr, Russell D. G.. 2000; Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature406:735–738
    [Google Scholar]
  22. Nemeria N., Yan Y., Zhang Z., Brown A. M., Arjunan P., Furey W., Guest J. R., Jordan F.. 2001; Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition. J Biol Chem276:45969–45978
    [Google Scholar]
  23. Nesbitt N. M., Yang X., Fontan P., Kolesnikova I., Smith I., Sampson N. S., Dubnau E.. 2009; A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol. Infect Immun78:275–282
    [Google Scholar]
  24. Sassetti C. M., Rubin E. J.. 2003; Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A100:12989–12994
    [Google Scholar]
  25. Sassetti C. M., Boyd D. H., Rubin E. J.. 2003; Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol48:77–84
    [Google Scholar]
  26. Shimakata T., Fujita Y., Kusaka T.. 1977; Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis. J Biochem82:725–732
    [Google Scholar]
  27. Shimakata T., Fujita Y., Kusaka T.. 1979; Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis. J Biochem86:1191–1198
    [Google Scholar]
  28. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H.. other authors 1991; New use of BCG for recombinant vaccines. Nature351:456–460
    [Google Scholar]
  29. Takayama K., Wang L., David H. L.. 1972; Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth, and viability of Mycobacterium tuberculosis. Antimicrob Agents Chemother2:29–35
    [Google Scholar]
  30. Velayati A. A., Masjedi M. R., Farnia P., Tabarsi P., Ghanavi J., Ziazarifi A. H., Hoffner S. E.. 2009; Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in Iran. Chest136:420–425
    [Google Scholar]
  31. Wheeler P. R., Ratledge C.. 1994; Metabolism of Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection and Control pp353–385 Edited by Bloom B. R. Washington, DC: American Society for Microbiology;
  32. Wheeler P. R., Bulmer K., Ratledge C.. 1990; Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?. J Gen Microbiol136:211–217
    [Google Scholar]
  33. Wheeler P. R., Bulmer K., Ratledge C.. 1991; Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens. J Gen Microbiol137:885–893
    [Google Scholar]
  34. WHO 2007; A World Free of TB.
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.038802-0
Loading
/content/journal/micro/10.1099/mic.0.038802-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error