Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of
            <i>Mycobacterium tuberculosis</i>

Takayama, K.; Lynn, Wang; David, Hugo L.

doi:10.1128/aac.2.1.29

Cited by 276 publications

(190 citation statements)

References 27 publications

(25 reference statements)

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“…Isoniazid was shown to inhibit the biosynthesis of mycolic acids (29), and the reductases involved in fatty-acid elongation isolated from Mycobacterium avium were shown to be selectively inhibited by the drug (17). More recent work has shown that the reductases are the targets of isoniazid inhibition in M. tuberculosis (1,25).…”

Section: Discussionmentioning

confidence: 99%

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

Daniel

Lee

et al. 2007

J Bacteriol

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The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (␣ 3 ) and AccD6 (␤ 6 ) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the ␣ 3 -␤ 6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the ␣ 3 -␤ 6 complex was inhibited by the subunit. The ␣ 3 -␤ 6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the ␤ 6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.Tuberculosis causes 2 million deaths each year, according to the World Health Organization. Mycobacterium tuberculosis, the pathogen that causes the disease, infects 8 million people each year and is one of the world's deadliest pathogens (9). The ongoing AIDS pandemic has developed a deadly synergy with tuberculosis, which is the leading cause of death among AIDS patients (2). Multidrug-resistant M. tuberculosis strains have been emerging rapidly (9), and the need for identifying novel drug targets in this pathogen has become urgent. The cell wall of M. tuberculosis is lipid enriched and acts as an impermeable barrier to many common broad-spectrum antibiotics (14).The first committed step of fatty-acid biosynthesis, which is the biotin-dependent carboxylation of acyl-coenzyme A (CoA) to produce malonyl-CoA and methylmalonyl-CoA, is catalyzed by the acyl-CoA carboxylase. The reaction consists of two catalytic steps, which involve the biotin carboxylase and the carboxyltransferase (8). In M. tuberculosis, the biotin carboxylation step is catalyzed by the ␣ subunit; there are three open reading frames (ORFs) that can encode the ␣ subunit (accA1 to -A3) in the genome. Carboxyl transfer is catalyzed by the ␤ subunit, and there are six ␤ subunits (accD1 to -D6) in the genome of the pathogen (6).Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the level...

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Section: Discussionmentioning

confidence: 99%

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

Daniel

Lee

et al. 2007

J Bacteriol

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“…Purification of the complex will be required to determine its precise nature. On the basis of the high sensitivity of the KasA-containing complex to alkaline pH (13), 5 it is tempting to hypothesize that the AcpM phosphopantetheine prosthetic group is directly involved in the covalent linking of AcpM and KasA. The phosphodiester group that links the prosthetic group to Ser-41 of AcpM 5 is the most chemically sensitive bond in the protein.…”

Section: Discussionmentioning

confidence: 99%

“…Middlebrook (3) was the first to carefully quantify the bactericidal activity of INH. Although Winder and Collins (4) demonstrated that INH inhibits mycolic acid biosynthesis, Takayama et al (5) demonstrated that inhibition of mycolic acid biosynthesis correlates with cell death. Takyama et al (6) further demonstrated that the inhibition of mycolic acid biosynthesis mediated by INH leads to the accumulation of a saturated C 26 fatty acid within 1 h of INH treatment.…”

Section: Isoniazid (Inh)mentioning

confidence: 99%

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

Journal of Biological Chemistry

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(6) further demonstrated that the inhibition of mycolic acid biosynthesis mediated by INH leads to the accumulation of a saturated C 26 fatty acid within 1 h of INH treatment. By using scanning electron microscopy, it was established that M. tuberculosis treated with INH undergoes a defined order of molecular events that leads to lysis 24 h after initiation of treatment, thus after approximately one cell generation (7). The events that lead from the inhibition of mycolic acid biosynthesis and the accumulation of a saturated C 26 fatty acid to cell lysis are still poorly understood. Moreover, there exists controversy as to which enzymes of the mycolic acid biosynthetic pathway are the actual targets of INH.The mode of action of INH appears to be rather complex and requires activation by the mycobacterial catalase-peroxidase KatG (8,9). Activation of the pro-drug leads to reactive radicals that exert a toxic effect on the tubercle bacillus (9 -11). Presumably activated INH inhibits one or more targets of the mycolic acid biosynthetic pathway which eventually leads to cell death. At least two enzymes have been identified as potential targets of activated INH, the enoyl-ACP reductase InhA (12) and the ␤-ketoacyl-ACP synthase KasA (13). The inhA gene was first identified by conferring co-resistance to INH and ethionamide (ETH) after overexpression in mycobacteria (12). In addition, mutations in inhA confer resistance to both INH and ETH in drug-resistant M. tuberculosis isolates (14 -17).

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“…Indeed, the cessation of mycolic acid synthesis is one of the primary effects of isoniazid (INH), 1 a front-line anti-tuberculosis drug (13). Various other compounds including ethionamide (14,15), isoxyl (15,16), and thiolactomycin (TLM) (17), have also been shown to inhibit mycolic acid synthesis.…”

mentioning

confidence: 99%

Purification and Biochemical Characterization of theMycobacterium tuberculosis β-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

Schaeffer¹,

Agnihotri²,

Volker³

et al. 2001

Journal of Biological Chemistry

135

130

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Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two ␤-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

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Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

Cited by 276 publications

References 27 publications

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

AccD6, a Member of the Fas II Locus, Is a Functional Carboxyltransferase Subunit of the Acyl-Coenzyme A Carboxylase in Mycobacterium tuberculosis

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Purification and Biochemical Characterization of theMycobacterium tuberculosis β-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

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