Tracking the Putative Biosynthetic Precursors of Oxygenated Mycolates of Mycobacterium tuberculosis
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m210501200
ISSN1083-351X
AutoresPremkumar Dinadayala, Françoise Laval, Catherine Raynaud, Anne Lemassu, Marie‐Antoinette Lanéelle, Gilbert Lanéelle, Mamadou Daffé,
Tópico(s)Infectious Diseases and Tuberculosis
ResumoDisruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630–637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of α-mycolates, composed of equal amounts of C76-C82 dicyclopropanated (α1) and of C77-C79 monoethylenic monocyclopropanated (α2) mycolates, the double bond being located at the "distal" position. In addition, small amounts ofcis-epoxymycolates, structurally related to α2-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene inMycobacterium smegmatis led to the lowering of diethylenic α mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis. Disruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630–637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of α-mycolates, composed of equal amounts of C76-C82 dicyclopropanated (α1) and of C77-C79 monoethylenic monocyclopropanated (α2) mycolates, the double bond being located at the "distal" position. In addition, small amounts ofcis-epoxymycolates, structurally related to α2-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene inMycobacterium smegmatis led to the lowering of diethylenic α mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis. fatty-acid synthase bacillus Calmette-Guérin gas chromatography mass spectrometry matrix-assisted laser desorption/ionization time-of-flight electron impact Mycolic acids, α-branched β-hydroxylated long-chain fatty acids (up to 90 carbon atoms), are the hallmark of theMycobacterium genus that comprises several human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively. These molecules represent major cell envelope components (40–60% of the cell dry weight) and are found covalently linked to the cell wall arabinogalactan or esterifying trehalose and glycerol; both types of mycolic acid-containing components are believed to play a crucial role in the structure and function of the mycobacterial cell envelope (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar, 2Goren M.B. Brennan P.J. Youmans G.P. Tuberculosis. Saunders Co., Philadelphia1979: 63-193Google Scholar, 3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Mycolic acids attached to the cell wall arabinogalactan are organized with other lipids to form an outer permeability barrier with an extremely low fluidity that confers an exceptional low permeability to mycobacteria and may explain their intrinsic resistance to many antibiotics (4Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1544) Google Scholar). Trehalose mycolates have been implicated in numerous biological functions related both to the physiology and virulence of Mycobacterium tuberculosis(3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar).Numerous studies have been and are currently devoted to the structures and biosynthesis of these acids, primarily because these substances are specific to the Mycobacterium genus, and their metabolism is the only clearly identified target inhibited by the major antitubercular drug, isoniazid (5Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (172) Google Scholar, 6Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (228) Google Scholar, 7Davidson L.A. Takayama K. Antimicrob. Agents Chemother. 1979; 16: 104-105Crossref PubMed Scopus (49) Google Scholar, 8Rozwarski D.A. Grant G.A. Barton D.H. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). With the re-emergence of tuberculosis infections caused by multidrug-resistant strains and the need for the development of new tuberculous drugs, deciphering the biosynthesis pathway leading to mycolates still represents a major objective of researchers. However, it remains that, despite the intensive efforts of biochemists over decades (3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 9Minnikin D.E. Ratledge C. Stanford J. The Biology of the Mycobacteria: Physiology, Identification and Classification. 1. Academic Press, London1982: 95-184Google Scholar, 10Lanéelle G. Acta Leprologica. 1989; 7: 65-73PubMed Google Scholar) and, more recently, the help of molecular genetic (11Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.G. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (447) Google Scholar, 12Kremer L. Baulard A.R. Besra G.S. Hartfull G.F. Jacobs W.R. Molecular Genetics of Mycobacteria. ASM Press, Washington, D. C.2000: 173-190Google Scholar), the biosynthesis pathway leading to mycolic acids is still poorly understood. Nevertheless, it is currently admitted that the two known mycobacterial fatty-acid synthases (FAS)1participate in the formation of all types of mycolates or their precursors. FAS-I, a synthase that has been shown to be a bimodal system, is necessary to produce C16, 18 and C22–26 saturated fatty acids, which may be either directly incorporated in mycolates as the α-branch chain or used as substrates of the elongation system, FAS-II. The finding that isoniazid strongly and specifically inhibits InhA, an enoyl-acyl carrier protein reductase that belongs to FAS-II (13Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1205) Google Scholar, 14Marrakchi H. Lanéelle G. Quémard A. Microbiology. 2000; 146: 289-296Crossref PubMed Scopus (181) Google Scholar), is consistent with the proposed biosynthetic pathway leading to the various types of mycolates.Mycolic acids occur usually in mycobacterial species as a mixture of various related molecules that differ from one another by the presence of chemical groups located on well-defined positions of their long methylene ("meromycolic") chain. In members of the M. tuberculosis complex, three types of mycolates are commonly encountered (15Daffé M. Lanéelle M.-A. Asselineau C. Lévy-Frébault V. David H.L. Ann. Microbiol. Inst. Pasteur. 1983; 134: 367-377Crossref Scopus (136) Google Scholar, 16Minnikin D.E. Minnikin S.M. Parlett J.H. Goodfellow M. Magnusson M. Arch. Microbiol. 1984; 139: 225-231Crossref PubMed Scopus (98) Google Scholar). The least polar mycolates, also called α-mycolates, are composed of C76-C82 fatty acids (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar) and contain two cis-cyclopropyl groups, at the so-called "proximal" and "distal" (relative to the carboxyl group) positions of the meromycolic chain (see Fig.1 A). The more polar "M" and "K" mycolates consist of C82-C89 substances (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar) and contain acis- or a trans (with an adjacent methyl group)-cyclopropyl group at the proximal position, and a methoxy- or a keto- group (with an adjacent methyl group) at the distal position (Fig. 1 A). These discrete structural variations in mycolates may be of crucial biological importance, because it has been shown that mutations resulting in the loss of these chemical functions profoundly modify the permeability of the cell envelope to solutes and severely affect the virulence of the mutant strains in experimental infections (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar, 18Yuan Y. Zhu Y. Crane D.D. Barry III, C.E. Mol. Microbiol. 1998; 29: 1449-1458Crossref PubMed Scopus (144) Google Scholar, 19Glickman M.S. Cox J.S. Jacobs W.R. Mol. Cell. 2000; 5: 717-727Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Accordingly, the enzymatic systems that introduce the chemical modifications in the mycolic acid chain merit special attention. Based on C-alkylation mechanisms (20Lederer E. Q. Rev. Chem. Soc. 1969; 23: 453-481Crossref Scopus (149) Google Scholar), a biosynthetic pathway that may explain the action of specific S-adenosylmethionine-dependent methyltransferases on ethylenic precursors leading to methyl branches and cyclopropanes in mycolates has been postulated (11Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.G. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (447) Google Scholar). Similar C-alkylation mechanisms have been proposed to explain the synthesis of keto- and methoxymycolic acids: the transformation of the distal double bond of a precursor into a secondary hydroxyl group with an adjacent methyl-branch using anS-adenosylmethionine-dependent methyltransferase coded by the gene mma4 (21Yuan Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (128) Google Scholar). A gene with the same function in Mycobacterium bovis BCG, first called cmaA(22Dubnau E. Lanéelle M.A. Soares S. Bénichou A. Vaz T. Promé D. Promé J.C. Daffé M. Quémard A. Mol. Microbiol. 1997; 23: 313-322Crossref PubMed Scopus (61) Google Scholar), when introduced in Mycobacterium smegmatis has been shown to confer to the latter organism the ability to produce ketomycolic acids. In addition, the transformant produced large amounts of hydroxymycolic acids with an adjacent methyl-branch, structurally related to the ketomycolic acids of M. bovis BCG. Trace amounts of these hydroxymycolic acids have been also detected in mycobacterial species producing keto- and/or methoxymycolates, further supporting the hypothesis that hydroxymycolic acids can be the precursors of both keto- and methoxymycolic acids (23Quémard A. Lanéelle M.A. Marrakchi H. Promé D. Dubnau E. Daffé M. Eur. J. Biochem. 1997; 250: 758-763Crossref PubMed Scopus (33) Google Scholar). Finally, a mutant strain of M. tuberculosis in which themma4 gene (thereafter called hma forhydroxymycolic acid) has been inactivated and shown to be devoid of both keto- and methoxymycolic acids (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar, 17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). Although this last experiment clearly established the biosynthetic relationships between the three oxygenated mycolates ofM. tuberculosis, the chemical structures of precursor molecules used by Hma to produce these oxygenated mycolates remain unknown. Interestingly, analysis of the mass spectra of the fatty acids from the mutant strain indicated the accumulation of new substances structurally related to α-mycolates and absent from the parent strain (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). In an attempt to identify these putative precursors of oxygenated mycolic acids of M. tuberculosis, a detailed analysis of the mycolic acid content of the hma knock-out mutant was performed using the combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, proton nuclear magnetic resonance (1H NMR) spectroscopy, and various chemical degradation techniques.DISCUSSIONWe have previously shown that the introduction of the genecmaA from M. bovis BCG Pasteur into M. smegmatis induced the production by the recipient strain of large amounts of methyl-branched hydroxymycolic acids and small amounts of ketomycolic acids (22Dubnau E. Lanéelle M.A. Soares S. Bénichou A. Vaz T. Promé D. Promé J.C. Daffé M. Quémard A. Mol. Microbiol. 1997; 23: 313-322Crossref PubMed Scopus (61) Google Scholar). Inactivation of the corresponding gene ofM. tuberculosis H37Rv, hma (or mma4), has resulted in the complete abolishment of the production of all the oxygenated mycolates normally present in M. tuberculosisH37Rv, namely the methoxy-, keto-, and hydroxymycolic acids (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar). It was thus concluded that the hma gene is involved in the synthesis of oxygenated mycolic acids and that hydroxymycolic acids are likely to be precursors of methoxy- and ketomycolates inM. tuberculosis. Interestingly, thehma-disrupted mutant strain was found to produce new types of mycolic acids absent from the parent strain, suggesting that these molecules may represent the precursors of the oxygenated mycolates ofM. tuberculosis (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). Accordingly, the present work was undertaken to elucidate the chemical structures of these molecules. Application of MALDI-TOF mass spectrometry, 1H NMR spectroscopy, and chemical degradation techniques to the analysis of the various purified subclasses of mycolates produced by the mutant established several facts. First, in the absence of the hmagene, the mutant strain synthesized as much mycolates as the parent strain, suggesting that the overall bacterial content in these molecules is important for the normal physiology of the bacilli. Second, both the mutant and parent strains synthesized similar amounts of dicyclopropanated α-mycolates exhibiting identical structures, indicating that the mutation did not affect the production of this type of mycolates. Third, the mutant produced large amounts of a new type of mycolates, namely a mixture of monoethylenic monocyclopropanated α-mycolates, and a tiny amount of cis-epoxy-containing monocyclopropanated mycolates; the ethylenic α-mycolates were synthesized throughout the various growth phases of the mutant, indicating that they did not arise from secondary reactions. Fourth, complementation of the mutant strain with the wild-type hmagene resulted in the disappearance of the new types of mycolates and the production by the complemented strain of all the types of mycolates of the parent strain. Finally, introduction of the hma gene in M. smegmatis led to the lowering of diethylenic α-mycolates of the recipient strain and the production of keto- and hydroxymycolates. Our results demonstrate that the observed changes in the structure of mycolates of the mutant are specifically due to the inactivation of the hma gene and raise some important questions regarding the biosynthesis of mycolic acids.One of the questions related to the biosynthesis of mycolic acids is the metabolic process that leads to the introduction of the specific oxygenated functions at the distal position of the molecules. Two classes of substrates could lead to oxygenated chemical functions after a C-methylation (20Lederer E. Q. Rev. Chem. Soc. 1969; 23: 453-481Crossref Scopus (149) Google Scholar, 35Asselineau C. Asselineau J. Lanéelle G. Lanéelle M.A. Prog. Lipid Res. 2002; 41: 501-523Crossref PubMed Scopus (68) Google Scholar), namely a keto group, leading to an α-methylated ketone (Fig.8 A), as known for menaquinones, or an ethylenic group (Fig. 8 B); in this latter case a water molecule should participate in the sulfonium-mediated addition mechanism, as proposed for Mma4 function (21Yuan Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (128) Google Scholar). Because only cis-ethylenic mycolate accumulates significantly in the hma-inactivated mutant, it is more attractive to postulate that the ethylenic distal double bond occurring in the new α-mycolates of the mutant strain is the substrate of Mma4/Hma. In the "ethylenic hypothesis" (Fig.8 B), the hydroxyl group formed after the methylation step of the ethylenic bond could be either transformed into a methoxyl group or oxidized into a ketone. In both cases, an oxidoreduction step would be necessary to obtain either the keto- or the methoxymycolic acids. Importantly, the values of m1 in the new α-mycolates are identical to those found in oxygenated mycolates of the parent strain and shorter than those observed in the α-mycolates of the parent strain (Fig. 1). Interestingly, the difference in chain lengths between α-mycolates and oxygenated mycolates have been previously observed (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar, 36Watanabe M. Aoyagi Y. Mitome H. Fujita T. Naoki H. Riddel M. Minnikin D.E. Microbiology. 2002; 148: 1881-1902Crossref PubMed Scopus (100) Google Scholar) and shown to be due to the specificity of the methyltransferases mainly with respect to the ω-end of the growing mero acyl chain (37Schroeder B.G. Barry III, C.E. Bioorganic Chem. 2001; 29: 164-177Crossref PubMed Scopus (15) Google Scholar).Figure 8Possible reaction mechanisms for the synthesis of oxygenated mycolic acids mediated by the Hma methyltransferase (adapted from Ref. 20Lederer E. Q. Rev. Chem. Soc. 1969; 23: 453-481Crossref Scopus (149) Google Scholar). A, the "ketone hypothesis"; B, the "ethylenic hypothesis."View Large Image Figure ViewerDownload (PPT)Based on the kinetics of production of α-mycolates and related oxygenated compounds in Mycobacterium microti (38Davidson L.A. Draper P. Minnikin D.E. J. Gen. Microbiol. 1982; 128: 823-828PubMed Google Scholar) and other mycobacterial species (39Lacave C. Lanéelle M.A. Daffé M. Montrozier H. Lanéelle G. Eur. J. Biochem. 1989; 181: 459-466Crossref PubMed Scopus (32) Google Scholar) on the one hand, and the chain lengths of these two classes of substances, on the other hand, it has been suggested that these molecules are synthesized by different enzymatic systems. Accordingly, the common precursor, if any, would not be a full "meromycolic" diethylenic compound that would then be modified by different methyltransferases, before or after the Claisen-type condensation step, to yield α- or/and oxygenated-mycolic acids (for a review see Ref. 3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). By showing that the hma-disrupted mutant produced dicyclopropanated α-mycolic acids as the parent strain and in comparable amounts, our data are consistent with the existence of one biosynthetic system devoted to the synthesis of α-mycolic acids and another machinery involved in the synthesis of oxygenated mycolic acids; only the latter one would be altered by the disruption of thehma gene. Both systems may contain a mixture of "core" enzymes whose activities are necessary for the synthesis of the meromycolic chain but may differ one from another by the association of additional specific enzymes involved in the introduction of various chemical groups in the meromycolic chain. The accumulation of large amounts of ethylenic α-mycolates in the hma-inactivated mutant, comparable to those of oxygenated mycolates of the parent strain, reinforces the hypothesis that ethylenic long chain fatty acid derivatives may be used for the biosynthesis of oxygenated mycolates in the parent and the hma-complemented strain. In the absence of the hma gene, these putative precursors would be used by the machinery originally devoted to the synthesis of oxygenated mycolates and yield distal-ethylenic compounds. The remaining unsolved question is the discrepancy between the chain lengths of the postulated precursors and those of the final products. Although the mutant produced C75–C83 monoethylenic monocyclopropanated α-mycolates (α2) and cis-epoxy monocyclopropanated mycolates, the parent and complemented strains synthesized C81–C89 methoxy- and ketomycolates, 4–6 carbon atoms longer than the α-mycolates (17Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar,26Watanabe M. Aoyagi Y. Riddel M. Minnikin D.E. Microbiology. 2001; 147: 1825-1837Crossref PubMed Scopus (123) Google Scholar). Examination of the detailed structures of mycolic acids, however, pointed to the observation that the additional carbons in oxygenated mycolates are not located between the methyl end and the oxygenated group but distributed in the other parts of the meromycolic chain. Accordingly, one can postulate that the introduction of an oxygenated function at this position induces enough hydrophilicity in the long carbon chain to slightly disturb the specificity toward chain lengths of the system in charge of the synthesis of the methylenic chain. As a consequence, the oxygenated groups should be introduced before completion of the meromycolic chain.Several genes coding for methyltransferases have been identified in the genome of M. tuberculosis (40Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Sulston J.E. Taylor K. Whitehead S. Barrel B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6445) Google Scholar). These enzymes are assumed to be responsible for the introduction of subtle variations in the mycolic acid structure, variations that may have profound effects on the physiology and virulence of the tubercle bacillus; for instance, the replacement of a cyclopropane ring by a double bond in α-mycolates esterifying trehalose totally abolishes the formation of cords typifying virulent tubercle bacilli and profoundly affects the virulence of the mutant strain (19Glickman M.S. Cox J.S. Jacobs W.R. Mol. Cell. 2000; 5: 717-727Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Similarly, the lack of production of keto- and methoxymycolates in M. tuberculosis results in both a change of the permeability and an attenuation of virulence of the mutant strain (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar). These observations, in addition to the fact that the biosynthesis pathway leading to mycolic acids is the target of the most effective antituberculous drug, isoniazid, suggest that methyltranferases may represent good targets for the development of new antituberculous drugs. In this respect, the identification of putative precursors of oxygenated mycolates should help in the design of substrate analogs that would be tested as inhibitors of the enzymatic activity of the Hma protein. The individual methyltransferases are not essential for the bacterial growth, but, because their crystal structures are highly similar (41Huang C.-C. Smith C.V. Glickman M.S. Jacobs W.R. Sacchettini J. C..J. Biol. Chem. 2002; 277: 11559-11569Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), the inhibitors of one enzyme may also abolish the activities of other methyltransferases, resulting in an additive effect that may be either lethal or bacteriostatic for the micro-organism. Mycolic acids, α-branched β-hydroxylated long-chain fatty acids (up to 90 carbon atoms), are the hallmark of theMycobacterium genus that comprises several human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively. These molecules represent major cell envelope components (40–60% of the cell dry weight) and are found covalently linked to the cell wall arabinogalactan or esterifying trehalose and glycerol; both types of mycolic acid-containing components are believed to play a crucial role in the structure and function of the mycobacterial cell envelope (1Dubnau E. Chan J. Raynaud C. Mohan V.P. Lanéelle M.A. Yu K. Quémard A. Smith I. Daffé M. Mol. Microbiol. 2000; 36: 630-637Crossref PubMed Scopus (253) Google Scholar, 2Goren M.B. Brennan P.J. Youmans G.P. Tuberculosis. Saunders Co., Philadelphia1979: 63-193Google Scholar, 3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Mycolic acids attached to the cell wall arabinogalactan are organized with other lipids to form an outer permeability barrier with an extremely low fluidity that confers an exceptional low permeability to mycobacteria and may explain their intrinsic resistance to many antibiotics (4Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1544) Google Scholar). Trehalose mycolates have been implicated in numerous biological functions related both to the physiology and virulence of Mycobacterium tuberculosis(3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Numerous studies have been and are currently devoted to the structures and biosynthesis of these acids, primarily because these substances are specific to the Mycobacterium genus, and their metabolism is the only clearly identified target inhibited by the major antitubercular drug, isoniazid (5Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (172) Google Scholar, 6Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (228) Google Scholar, 7Davidson L.A. Takayama K. Antimicrob. Agents Chemother. 1979; 16: 104-105Crossref PubMed Scopus (49) Google Scholar, 8Rozwarski D.A. Grant G.A. Barton D.H. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). With the re-emergence of tuberculosis infections caused by multidrug-resistant strains and the need for the development of new tuberculous drugs, deciphering the biosynthesis pathway leading to mycolates still represents a major objective of researchers. However, it remains that, despite the intensive efforts of biochemists over decades (3Daffé M. Draper P. Adv. Microbiol. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 9Minnikin D.E. Ratledge C. Stanford J. The Biology of the Mycobacteria: Physiology, Identification and Classification. 1. Academic Press, London1982: 95-184Google Scholar, 10Lanéelle G. Acta Leprologica. 1989; 7: 65-73PubMed Google Scholar) and, more recently, the help of molecular genetic (11Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.G. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Crossref PubMed Scopus (447) Google Scholar, 12Kremer L. Baulard A.R. Besra G.S. Hartfull G.F. Jacobs W.R. Molecular Genetics of Mycobacteria. ASM Press, Washington, D. C.2000: 173-190Google Scholar), the biosynthesis pathway leading to mycolic acids is still poorly understood. Nevertheless, it is currently admitted that the two known mycobacterial fatty-acid synthases (FAS)1participate in the formation of all types of mycolates or their precursors. FAS-I, a synthase th
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