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

Schaeffer, Merrill L.; Agnihotri, Gautam; Volker, Craig; Kallender, Howard; Brennan, Patrick J.; Lonsdale, John T.

doi:10.1074/jbc.m108903200

Cited by 135 publications

(139 citation statements)

References 43 publications

(43 reference statements)

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“…The FAS II complex in M. tuberculosis is located in a genetic locus that also contains a lone, uncharacterized acyl-CoA carboxylase carboxyltransferase subunit, accD6 (␤ 6 ) (6), which could provide malonylCoA to the ␤-ketoacyl-ACP synthases and to FAS I. MalonylCoA is converted to malonyl-ACP by the malonyl-CoA-ACP transacylase (FabD) (19) and is utilized by the ␤-ketoacyl-ACP synthases (KasA/KasB) (5,18,27) in successive reactions with the other enzymes of the FAS II complex to produce the meromycolic acids. Protein-protein interaction analyses have shown that FabD is a structural component of the FAS II complex (30) and that KasA, KasB, InhA, MabA (FabG1), and FabH (␤-ketoacyl-ACP synthase III) interact with each other (31).…”

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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“…The upregulated protein b-ketoacyl-ACP synthase (KasB, Rv2246) is, together with KasA, involved in the synthesis of mycolic acids (Schaeffer et al, 2001). KasA has been shown to specifically elongate palmitoyl-CoA to monounsaturated fatty acids averaging 40 carbons in length and overproduction of KasB in the presence of KasA leads to the production of even longer chains.…”

Section: Cell Wallmentioning

confidence: 99%

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions

Starck

Källenius

Marklund³

et al. 2004

Microbiology

135

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Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the a-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), b-ketoacyl-ACP synthase (KasB), L-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy. INTRODUCTIONHuman tuberculosis (TB) is caused by an intracellular pathogen, Mycobacterium tuberculosis, which causes both latent and acute illness. Approximately eight million active cases are reported annually with 2 million deaths. In addition, about one-third of the global population is estimated to suffer from latent tuberculosis (Dye et al., 1999), which can be reactivated even after several decades. The fact that the bacilli can shift into a dormant state is of therapeutic importance, since this dormant state induces resistance to the two most important TB-drugs, rifampicin and isoniazide (Wayne & Sramek, 1994). One of the most challenging problems in TB research is to reveal the mechanisms behind latency.Latency is believed to involve a non-replicating persistence of mycobacteria or a very slow growth. One important condition believed to contribute to latency is reduced access to oxygen. M. tuberculosis is generally regarded as a strictly aerobic bacillus, although it can survive for a long time in a micro-aerophilic environment as long as the shift is not too abrupt (Wayne & Hayes, 1996). An in vitro model to study this persistent state is the so-called Wayne dormancy model, in which the bacteria downregulate their metabolism due to reduced access to oxygen (Wayne & Hayes, 1996). Another dormancy model utilizes nutrient starvation to induce a state where M. tuberculosis arrests growth and decreases its respiration rate (Betts et al., 2002). Recently it was demonstrated that inhibition of respiration by nitric oxide might induce a dormant state in M. tuberculosis . One should recognize, however, that the evidence linking in vitro dormant states of M. tuberculosis to human latent infection still remains circumstantial.The vaccine strain Mycobacterium bovis BCG has been reported to occasionally persist in a latent state in ...

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“…CER is a drug that is produced naturally by Cephalosporium caerulens and exhibits potent activity against a wide variety of yeast, fungi, and bacteria (53) through inhibition of both FAS-I and FAS-II (43,54). Several studies (34,43,55,56) have demonstrated that it specifically targets ␤-ketoacyl-ACP synthases by covalently attaching to the active site cysteine. CER is known to inhibit KasA activity in vitro (34,56).…”

Section: Induction Of the Kasa-containing Complex By Inha-inhibitory mentioning

confidence: 99%

“…Several studies (34,43,55,56) have demonstrated that it specifically targets ␤-ketoacyl-ACP synthases by covalently attaching to the active site cysteine. CER is known to inhibit KasA activity in vitro (34,56). However, this drug was not able to inhibit InhA activity in vitro (Table IV), and the MIC of CER for a strain overproducing InhA was found to be comparable with that for the control strain (Table III), confirming that InhA is not inhibited by CER.…”

Section: Induction Of the Kasa-containing Complex By Inha-inhibitory mentioning

confidence: 99%

“…that has been shown to inhibit mycobacterial growth (41) by targeting both ␤-ketoacyl-ACP synthases KasA and KasB (27,56). M. bovis BCG was chosen because it is more sensitive to ISO and TLM than M. smegmatis, the MIC value of ISO for M. smegmatis is 600 g/ml (Table III), and it corresponds to 0.5 g/ml for M. bovis BCG (57).…”

Section: Induction Of the Kasa-containing Complex By Inha-inhibitory mentioning

confidence: 99%

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

Cited by 135 publications

References 43 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

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions

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

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