Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development.

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for de novo synthesis of branched-chain amino acids. The genome sequence of Mycobacterium tuberculosis revealed genes encoding four catalytic subunits, ilvB1 (Rv3003c), ilvB2 (Rv3470c), ilvG (Rv1820) and ilvX (Rv3509c), and one regulatory subunit, ilvN (Rv3002c), of AHAS. All these genes were found to be expressed in M. tuberculosis growing in vitro. Each AHAS subunit gene was cloned and expressed in Escherichia coli. 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 M. tuberculosis, expression of AHAS genes was analysed in different physiological conditions. ilvB1, ilvB2 and ilvG were differentially expressed. The role of ilvB1 in persistence is known, but the upregulation of ilvB2 and ilvG in extended stationary phase, ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of M. tuberculosis by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.

[1]  V. Srivastava,et al.  Identification of genes of Mycobacterium tuberculosis upregulated during anaerobic persistence by fluorescence and kanamycin resistance selection. , 2008, Tuberculosis.

[2]  B. Miflin.  Cooperative feedback control of barley acetohydroxyacid synthetase by leucine, isoleucine, and valine. , 1971, Archives of biochemistry and biophysics.

[3]  F. Störmer.  The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. I. Kinetic studies. , 1968, The Journal of biological chemistry.

[4]  N. Najimudin,et al.  Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin , 1993, Journal of bacteriology.

[5]  F. Störmer.  The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. II. Evidence that it is not a flavoprotein. , 1968, The Journal of biological chemistry.

[6]  Dong-Eun Kim,et al.  Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors. , 2007, Archives of biochemistry and biophysics.

[7]  F. C. Størmer,et al.  Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes , 1975, Journal of bacteriology.

[8]  D. Goodlett,et al.  ICAT-based comparative proteomic analysis of non-replicating persistent Mycobacterium tuberculosis. , 2006, Tuberculosis.

[9]  J. Betts,et al.  Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling , 2002, Molecular microbiology.

[10]  Moon-Young Yoon,et al.  Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library , 2005, FEBS letters.

[11]  J. Gerberding,et al.  Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs--worldwide, 2000-2004. , 2006, MMWR. Morbidity and mortality weekly report.

[12]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[13]  V. Subbulakshmi,et al.  Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice. , 2009, Microbiology.

[14]  J. Schloss,et al.  Inhibitors of branched-chain amino acid biosynthesis as potential antituberculosis agents. , 1998, The Journal of antimicrobial chemotherapy.

[15]  M. Penttilä,et al.  Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes , 1993, Journal of bacteriology.

[16]  N. Kosaric,et al.  The Microbial Production of 2,3-Butanediol , 1987 .

[17]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[18]  Z. Barak,et al.  Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones. , 2003, Biochimica et biophysica acta.

[19]  R. P. Wagner,et al.  An acetohydroxy acid synthetase from Neurospora crassa. , 1968, Archives of biochemistry and biophysics.

[20]  W B Whitman,et al.  Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus , 1994, Journal of bacteriology.

[21]  W. Jacobs,et al.  Attenuation of and Protection Induced by a Leucine Auxotroph of Mycobacterium tuberculosis , 2000, Infection and Immunity.

[22]  J. Snoep,et al.  Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis , 1992, Journal of bacteriology.

[23]  L. Wayne,et al.  An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence , 1996, Infection and immunity.

[24]  T. Henkin,et al.  Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis , 1993, Molecular microbiology.

[25]  F. C. Størmer.  Evidence for regulation of Aerobacter aerogenes pH 6 acetolactate-forming enzyme by acetate ion. , 1977, Biochemical and biophysical research communications.

[26]  A. Böck,et al.  Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena , 1995, Journal of bacteriology.

[27]  T. Montville,et al.  Conversion of Pyruvate to Acetoin Helps To Maintain pH Homeostasis in Lactobacillus plantarum , 1992, Applied and environmental microbiology.

[28]  G. Bancroft,et al.  Characterization of Auxotrophic Mutants ofMycobacterium tuberculosis and Their Potential as Vaccine Candidates , 2001, Infection and Immunity.

[29]  C. Locht,et al.  Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection. , 2007, Microbiology.

[30]  Parissa Farnia,et al.  Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. , 2009, Chest.

[31]  T. Feltwell,et al.  Erratum: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[32]  E. Freese,et al.  Control of metabolite secretion in Bacillus subtilis. , 1973, Journal of general microbiology.

[33]  W. Jacobs,et al.  Auxotrophic vaccines for tuberculosis , 1996, Nature Medicine.

[34]  T. Weisbrod,et al.  In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis , 1995, Infection and immunity.

[35]  J. Yang,et al.  Purification and characterization of the valine sensitive acetolactate synthase from Serratia marcescens ATCC 25419. , 1993, Biochimica et biophysica acta.

[36]  W. D. Holtzclaw,et al.  Degradative acetolactate synthase of Bacillus subtilis: purification and properties , 1975, Journal of bacteriology.