Thiolactomycin, a new antibiotic. III. In vitro antibacterial activity.

Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.Diacylglycerol kinases (DagKs) 5 are key enzymes in phospholipid metabolism that function to reintroduce DAG formed from the breakdown of phospholipids into the biosynthetic pathway. In Escherichia coli, PtdGro is degraded to DAG by the transfer of the sn-glycerol-1-P head group to membrane-derived oligosaccharides

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“…Aliquots (150 l) were flash-frozen in liquid nitrogen and stored at Ϫ80°C. Protein was measured by the Bradford method (25).…”

Section: Methodsmentioning

confidence: 99%

Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis

Jerga

Schujman

et al. 2007

Journal of Biological Chemistry

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“…The initiation condensing enzyme, FabH, and elongation condensing enzymes, FabF/B, are essential components of this biosynthetic pathway (14)(15)(16)(17) and consequently are highly conserved among key pathogens. Although no drugs targeting condensing enzymes are used in the clinic, two inhibitors, cerulenin (18) and thiolactomycin (19), with poor antibacterial activities were discovered more than two decades ago. Cerulenin selectively targets FabF/B forming a covalent bond with the catalytic cysteine in the active site with its tail occupying the long hydrophobic cavity that normally contains the growing acyl chain of the natural substrate (20,21).…”

mentioning

confidence: 99%

Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties

Wang

Kodali²,

Lee³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

371

366

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Emergence of bacterial resistance is a major issue for all classes of antibiotics; therefore, the identification of new classes is critically needed. Recently we reported the discovery of platensimycin by screening natural product extracts using a target-based whole-cell strategy with antisense silencing technology in concert with cell free biochemical validations. Continued screening efforts led to the discovery of platencin, a novel natural product that is chemically and biologically related but different from platensimycin. Platencin exhibits a broad-spectrum Gram-positive antibacterial activity through inhibition of fatty acid biosynthesis. It does not exhibit cross-resistance to key antibiotic resistant strains tested, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, and vancomycin-resistant Enterococci. Platencin shows potent in vivo efficacy without any observed toxicity. It targets two essential proteins, ␤-ketoacyl-[acyl carrier protein (ACP)] synthase II (FabF) and III (FabH) with IC 50 values of 1.95 and 3.91 g/ml, respectively, whereas platensimycin targets only FabF (IC 50 ‫؍‬ 0.13 g/ml) in S. aureus, emphasizing the fact that more antibiotics with novel structures and new modes of action can be discovered by using this antisense differential sensitivity wholecell screening paradigm.platensimycin ͉ natural product ͉ thiolactomycin ͉ antisense ͉ fatty acid synthesis

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“…Thiolactomycin (TLM), a natural product thiolactone isolated from Nocardia sp., is a reversible KAS enzyme inhibitor (14,16,23,24) with activity against both Gram-positive and Gram-negative bacteria (25,26) as well as MTB (MIC 62.5 M) (27,28). Although TLM has also been reported to inhibit the human FAS-I enzyme (29), the low toxicity and relatively low affinity of TLM for FAS-I (IC 50 100 M) make it an attractive lead compound for antimicrobial drug discovery (30).…”

mentioning

confidence: 99%

Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

Machutta

Bommineni

Luckner

et al. 2010

Journal of Biological Chemistry

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Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the ␤-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acylenzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices ␣5 and ␣6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100 -200 g/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

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Thiolactomycin, a new antibiotic. III. In vitro antibacterial activity.

Cited by 116 publications

References 2 publications

Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis

Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis

Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties

Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

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