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Abstract

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In , DHAD is encoded by gene , and it shares 40 % amino acid sequence identity and conserved motifs with DHAD of encoded by . In this study, was overexpressed in and the resultant protein was characterized as a homodimer (∼155 kDa). Functional characterization of was established by biochemical testing and by genetic complementation of an intron-disrupted -auxotrophic mutant of to prototrophy. Growth of , BL21(DE3) and recombinant BL21(DE3) Δ carrying was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of by NO probably inhibited bacterial growth. The role of in was elucidated by antisense and sense RNA constructs. Growth of transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of was observed during the early exponential phase of growth, under acid stress and , suggesting that has a role in the survival of during normal and stress conditions. It may be concluded that the DHAD encoded by is essential for the survival of and could be a potential drug/vaccine target, as it is absent in mammals.

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2011-01-01
2019-10-19
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References

  1. Akhtar, P., Srivastava, S., Srivastava, A., Srivastava, M., Srivastava, B. S. & Srivastava, R. ( 2006; ). Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice. Microbes Infect 8, 2855–2862.[CrossRef]
    [Google Scholar]
  2. Arfin, S. M. ( 1969; ). Evidence for an enol intermediate in the enzymatic conversion of α,β-dihydroxyisovalerate to α-ketoisovalerate. J Biol Chem 244, 2250–2251.
    [Google Scholar]
  3. Armstrong, F. B., Muller, U. S., Reary, J. B., Whitehouse, D. & Croute, D. H. ( 1977; ). Stereoselectivity and stereospecificity of the α,β-dihydroxyacid dehydratase from Salmonella typhimurium. Biochim Biophys Acta 498, 282–293.[CrossRef]
    [Google Scholar]
  4. Bange, F. C., Brown, A. M. & Jacobs, W. R., Jr ( 1996; ). Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages. Infect Immun 64, 1794–1799.
    [Google Scholar]
  5. 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]
  6. Chan, J., Xing, Y., Magliozzo, R. S. & Bloom, B. R. ( 1992; ). Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med 175, 1111–1122.[CrossRef]
    [Google Scholar]
  7. Cioffi, E. A., Shaw, K. J., Bailey, W. F. & Berg, C. M. ( 1980; ). Improved synthesis of the sodium salt of dl-alpha,beta-dihydroxyisovaleric acid. Anal Biochem 104, 485–488.[CrossRef]
    [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. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  9. Edwards, K. M., Cynamon, M. H., Voladri, R. K., Hager, C. C., DeStefano, M. S., Tham, K. T., Lakey, D. L., Bochan, M. R. & Kernodle, D. S. ( 2001; ). Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis. Am J Respir Crit Care Med 164, 2213–2219.[CrossRef]
    [Google Scholar]
  10. Flint, D. H., Emptage, M. H., Finnegan, M. G., Fu, W. & Johnson, M. K. ( 1993a; ). The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase. J Biol Chem 268, 14732–14742.
    [Google Scholar]
  11. Flint, D. H., Smyk-Randall, E., Tuminello, J. F., Draczynska-Lusiak, B. & Brown, O. R. ( 1993b; ). The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated. J Biol Chem 268, 25547–25552.
    [Google Scholar]
  12. 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]
  13. 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]
  14. Hickey, M. J., Arain, T. M., Shawar, R. M., Humble, D. J., Langhorne, M. H., Morgenroth, J. N. & Stover, C. K. ( 1996; ). Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice. Antimicrob Agents Chemother 40, 400–407.
    [Google Scholar]
  15. Hofmann, M. A. & Brian, D. ( 1991; ). Sequencing PCR DNA amplified directly from a bacterial colony. Biotechniques 11, 30–31.
    [Google Scholar]
  16. 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]
  17. Hyduke, D. R., Jarboe, L. R., Tran, L. M., Chou, K. J. & Liao, J. C. ( 2007; ). Integrated network analysis identifies nitric oxide response networks and dihydroxyacid dehydratase as a crucial target in Escherichia coli. Proc Natl Acad Sci U S A 104, 8484–8489.[CrossRef]
    [Google Scholar]
  18. Ignarro, L. J. ( 2000; ). Nitric Oxide: Biology and Pathobiology. San Diego, CA. : Academic Press.
    [Google Scholar]
  19. Kanamori, M. & Wixom, R. L. ( 1963; ). Studies in valine biosynthesis. V. Characteristics of the purified dihydroxyacid dehydratase from spinach leaves. J Biol Chem 238, 998–1005.
    [Google Scholar]
  20. Kim, S. & Lee, S. B. ( 2006; ). Catalytic promiscuity in dihydroxy-acid dehydratase from the thermoacidophilic archaeon Sulfolobus solfataricus. J Biochem 139, 591–596.[CrossRef]
    [Google Scholar]
  21. Lin, G., Li, D., de Carvalho, L. P., Deng, H., Tao, H., Vogt, G., Wu, K., Schneider, J., Chidawanyika, T. & other authors ( 2009; ). Inhibitors selective for mycobacterial versus human proteasomes. Nature 461, 621–626.[CrossRef]
    [Google Scholar]
  22. 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]
  23. Pablos-Méndez, A., Raviglione, M. C., Laszlo, A., Binkin, N., Rieder, H. L., Bustreo, F., Cohn, D. L., Lambregts-van Weezenbeek, C. S., Kim, S. J. & other authors ( 1998; ). Global surveillance for antituberculosis-drugs resistance. 1994–1997. N Engl J Med 338, 1641–1649.[CrossRef]
    [Google Scholar]
  24. Pavelka, M. S., Jr, Chen, B., Kelley, C. L., Collins, F. M. & Jacobs Jr, W. R., Jr ( 2003; ). Vaccine efficacy of a lysine auxotroph of Mycobacterium tuberculosis. Infect Immun 71, 4190–4192.[CrossRef]
    [Google Scholar]
  25. Perutka, J., Wang, W., Goerlitz, D. & Lambowitz, A. M. ( 2004; ). Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol 336, 421–439.[CrossRef]
    [Google Scholar]
  26. Ren, B., Zhang, N., Yang, J. & Ding, H. ( 2008; ). Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli. Mol Microbiol 70, 953–964.
    [Google Scholar]
  27. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. ( 2001; ). Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A 98, 12712–12717.[CrossRef]
    [Google Scholar]
  28. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. ( 2003; ). Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48, 77–84.[CrossRef]
    [Google Scholar]
  29. Singh, V., Chandra, D., Srivastava, B. S. & Srivastava, R. ( 2011; ). Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development. Microbiology 157, 29–37.[CrossRef]
    [Google Scholar]
  30. 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]
  31. Spiro, S. ( 2007; ). Regulators of bacterial responses to nitric oxide. FEMS Microbiol Rev 31, 193–211.[CrossRef]
    [Google Scholar]
  32. Wilson, T., de Lisle, G. W., Marcinkeviciene, J. A., Blanchard, J. S. & Collins, D. M. ( 1998; ). Antisense RNA to ahpC, an oxidative stress defence gene involved in isoniazid resistance, indicates that AhpC of Mycobacterium bovis has virulence properties. Microbiology 144, 2687–2695.[CrossRef]
    [Google Scholar]
  33. Wu, S., Howard, S. T., Lakey, D. L., Kipnis, A., Samten, B., Safi, H., Gruppo, V., Wizel, B., Shams, H. & other authors ( 2004; ). The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo. Mol Microbiol 51, 1551–1562.[CrossRef]
    [Google Scholar]
  34. Xing, R. Y. & Whitman, W. ( 1991; ). Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp. J Bacteriol 173, 2086–2092.
    [Google Scholar]
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vol. , part 1, pp. 38 - 46

[ PDF, 273 kb], including: Sequences of oligonucleotide primers used in the study CLUSTAL W alignment of the predicted amino acid sequence of IlvD proteins from mycobacteria and



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