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Abstract

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for synthesis of branched-chain amino acids. The genome sequence of revealed genes encoding four catalytic subunits, (), (), () and (), and one regulatory subunit, (), of AHAS. All these genes were found to be expressed in growing . Each AHAS subunit gene was cloned and expressed in . AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology of , expression of AHAS genes was analysed in different physiological conditions. , and were differentially expressed. The role of in persistence is known, but the upregulation of and in extended stationary phase, , and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.

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2011-01-01
2019-10-19
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References

  1. Awasthy, D., Gaonkar, S., Shandil, R. K., Yadav, R., Bharath, S., Marcel, N., Subbulakshmi, V. & Sharma, U. ( 2009; ). Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice. Microbiology 155, 2978–2987.[CrossRef]
    [Google Scholar]
  2. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. ( 2002; ). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717–731.[CrossRef]
    [Google Scholar]
  3. Blomqvist, K., Nikkola, M., Lehtovaara, P., Suihko, M. L., Airaksinen, U., Stråby, K. B., Knowles, J. K. & Penttilä, M. E. ( 1993; ). Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes. J Bacteriol 175, 1392–1404.
    [Google Scholar]
  4. Bradford, M. M., McRorie, R. A. & Williams, W. L. ( 1976; ). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  5. Centers for Disease Control and Prevention ( 2006; ). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs – worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 55, 301–305.
    [Google Scholar]
  6. Cho, S. H., Goodlett, D. & Franzblau, S. ( 2006; ). ICAT-based comparative proteomic analysis of non-replicating persistent Mycobacterium tuberculosis. Tuberculosis (Edinb) 86, 445–460.[CrossRef]
    [Google Scholar]
  7. Choi, K. J., Yu, Y. G., Hahn, H. G., Choi, J. D. & Yoon, M. Y. ( 2005; ). Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library. FEBS Lett 579, 4903–4910.[CrossRef]
    [Google Scholar]
  8. Choi, K. J., Noh, K. M., Kim, D. E., Ha, B. H., Kim, E. E. & Yoon, M. Y. ( 2007; ). Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors. Arch Biochem Biophys 466, 24–30.[CrossRef]
    [Google Scholar]
  9. 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. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  10. Grandoni, J. A., Marta, P. T. & Schloss, J. V. ( 1998; ). Inhibitors of branched-chain amino acid biosynthesis as potential antituberculosis agents. J Antimicrob Chemother 42, 475–482.[CrossRef]
    [Google Scholar]
  11. Grundy, F. J., Waters, D. A., Takova, T. Y. & Henkin, T. M. ( 1993; ). Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis. Mol Microbiol 10, 259–271.[CrossRef]
    [Google Scholar]
  12. Guleria, I., Teitelbaum, R., McAdam, R. A., Kalpana, G., Jacobs, W. R., Jr & Bloom, B. R. ( 1996; ). Auxotrophic vaccines for tuberculosis. Nat Med 2, 334–337.[CrossRef]
    [Google Scholar]
  13. Holtzclaw, W. D. & Chapman, L. F. ( 1975; ). Degradative acetolactate synthase of Bacillus subtilis: purification and properties. J Bacteriol 121, 917–922.
    [Google Scholar]
  14. Hondalus, M. K., Bardarov, S., Russell, R., Chan, J., Jacobs, W. R., Jr & Bloom, B. R. ( 2000; ). Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 68, 2888–2898.[CrossRef]
    [Google Scholar]
  15. Johansen, L., Bryn, K. & Störmer, F. C. ( 1975; ). Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes. J Bacteriol 123, 1124–1130.
    [Google Scholar]
  16. Kuwana, H., Caroline, D. F., Harding, R. W. & Wagner, R. P. ( 1968; ). An acetohydroxy acid synthetase from Neurospora crassa. Arch Biochem Biophys 128, 184–193.[CrossRef]
    [Google Scholar]
  17. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  18. Magee, R. J. & Kosaric, N. ( 1987; ). The microbial production of 2,3-butanediol. Adv Appl Microbiol 32, 89–161.
    [Google Scholar]
  19. Mayer, D., Schlensog, V. & Böck, A. ( 1995; ). Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena. J Bacteriol 177, 5261–5269.
    [Google Scholar]
  20. McAdam, R. A., Weisbrod, T. R., Martin, J., Scuderi, J. D., Brown, A. M., Cirillo, J. D., Bloom, B. R. & Jacobs, W. R., Jr ( 1995; ). In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infect Immun 63, 1004–1012.
    [Google Scholar]
  21. Miflin, B. J. ( 1971; ). Cooperative feedback control of barley acetohydroxyacid synthetase by leucine, isoleucine, and valine. Arch Biochem Biophys 146, 542–550.[CrossRef]
    [Google Scholar]
  22. Renna, M. C., Najimudin, N., Winik, L. R. & Zahler, S. A. ( 1993; ). Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J Bacteriol 175, 3863–3875.
    [Google Scholar]
  23. Sambandamurthy, V. K., Wang, X., Chen, B., Russell, R. G., Derrick, S., Collins, F. M., Morris, S. L. & Jacobs, W. R., Jr ( 2002; ). A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nat Med 8, 1171–1174.[CrossRef]
    [Google Scholar]
  24. Saxena, A., Srivastava, V., Srivastava, R. & Srivastava, B. S. ( 2008; ). Identification of genes of Mycobacterium tuberculosis upregulated during anaerobic persistence by fluorescence and kanamycin resistance selection. Tuberculosis (Edinb) 88, 518–525.[CrossRef]
    [Google Scholar]
  25. Smith, D. A., Parish, T., Stoker, N. G. & Bancroft, G. J. ( 2001; ). Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 69, 1142–1150.[CrossRef]
    [Google Scholar]
  26. Snoep, J. L., Teixeira de Mattos, M. J., Starrenburg, M. J. & Hugenholtz, J. ( 1992; ). Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis. J Bacteriol 174, 4838–4841.
    [Google Scholar]
  27. Speck, E. L. & Freese, E. ( 1973; ). Control of metabolite secretion in Bacillus subtilis. J Gen Microbiol 78, 261–275.[CrossRef]
    [Google Scholar]
  28. Srivastava, V., Rouanet, C., Srivastava, R., Ramalingam, B., Locht, C. & Srivastava, B. S. ( 2007; ). Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection. Microbiology 153, 659–666.[CrossRef]
    [Google Scholar]
  29. Störmer, F. C. ( 1968a; ). The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. I. Kinetic studies. J Biol Chem 243, 3735–3739.
    [Google Scholar]
  30. Störmer, F. C. ( 1968b; ). The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. II. Evidence that it is not a flavoprotein. J Biol Chem 243, 3740–3741.
    [Google Scholar]
  31. Störmer, F. C. ( 1977; ). Evidence for regulation of Aerobacter aerogenes pH 6 acetolactate-forming enzyme by acetate ion. Biochem Biophys Res Commun 74, 898–902.[CrossRef]
    [Google Scholar]
  32. Tsau, J.-L., Guffanti, A. A. & Montville, T. J. ( 1992; ). Conversion of pyruvate to acetoin helps to maintain pH homeostasis in Lactobacillus plantarum. Appl Environ Microbiol 58, 891–894.
    [Google Scholar]
  33. 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. Chest 136, 420–425.[CrossRef]
    [Google Scholar]
  34. Wayne, L. G. & Hayes, L. G. ( 1996; ). An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64, 2062–2069.
    [Google Scholar]
  35. WHO ( 2009; ). Global tuberculosis control: epidemiology, strategy, financing: WHO Report (Publication no. WHO/HTM/TB/2009.411). Geneva: World Health Organization.
  36. Xing, R. & Whitman, W. B. ( 1994; ). Purification and properties of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus. J Bacteriol 176, 1207–1213.
    [Google Scholar]
  37. Yang, J. H. & Kim, S. S. ( 1993; ). Purification and characterization of the valine sensitive acetolactate synthase from Serratia marcescens ATCC 25419. Biochim Biophys Acta 1157, 178–184.[CrossRef]
    [Google Scholar]
  38. Zohar, Y., Einav, M., Chipman, D. M. & Barak, Z. ( 2003; ). Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones. Biochim Biophys Acta 1649, 97–105.[CrossRef]
    [Google Scholar]
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vol. , part 1, pp. 29 - 37

[ PDF, 330 kb], including: Protein expression strategy for recombinant AHAS proteins Purification strategy for recombinant AHAS proteins SDS-PAGE analysis of recombinant AHAS proteins



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