Thiolactomycin, a new antibiotic. I. Taxonomy of the producing organism, fermentation and biological properties.

Oishi, Hideo; Noto, Takao; Sasaki, Hiroshi; Suzuki, Koji; Hayashi, Toshiaki; Okazaki, Hiroshi; Ando, Kiyoshi; Sawada, Mikio

doi:10.7164/antibiotics.35.391

Cited by 153 publications

(81 citation statements)

References 15 publications

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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).…”

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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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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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“…,B-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (17). The role of this third condensing enzyme remains to be established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the total rate of fatty acid production.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (yeast and mammalian) fatty acid synthases (14,15,25,26,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23).…”

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Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12

1992

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Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional I8-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabBIS(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented thefabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM-resistant fatty acid biosynthesis in vivo and in vitro. These data show that ,I-ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance.The 3-ketoacyl-acyl carrier protein (ACP) synthases are key regulators of dissociated (type II) fatty acid synthase systems typified by the Escherichia coli system (for reviews, see references 5 and 28). ,-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP, and mutants (fabB) lacking synthase I activity are unable to synthesize either palmitoleic or cis-vaccenic acid and require supplementation with unsaturated fatty acids for growth (6, 30). 13-Ketoacyl-ACP synthase II is responsible for the temperature-dependent regulation of fatty acid composition (for a review, see reference 7). Mutants (fabF) lacking synthase II activity are deficient in cis-vaccenic acid but grow normally (11,12). ,B-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (17). The role of this third condensing enzyme remains to be established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the total rate of fatty acid production.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (yeast and mammalian) fatty acid synthases (14,15,25,26,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23). Understanding the mechanism of TLM ac...

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“…This potential is further underlined by the potency of the classic drugs isoniazid and triclosan, both inhibiting the ER step of bacterial fatty acid biosynthesis (6,7). Several inhibitors targeting the ketoacyl synthase (KS) step of the FAS cycle have also been described, including cerulenin (CER) (8), thiolactomycin (TLM) (9), and the recently discovered platensimycin (PLM) (10). The polyketide CER inhibits both FAS type I and II KS enzymes, by covalent modification of the active site cysteine and by occupying the long acyl-binding pocket (11,12).…”

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Inhibition of the fungal fatty acid synthase type I multienzyme complex

Johansson

Wiltschi

Kumari

et al. 2008

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

118

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Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413-K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.cerulenin ͉ platensimycin ͉ thiolactomycin ͉ fatty acid synthesis ͉ yeast T hree different schemes for de novo synthesis of fatty acids are found in nature. Eukaryotes and advanced prokaryotes generally use the type I fatty acid synthase system (FAS I), composed of complexes of large multifunctional enzymes. Bacteria, in contrast, use the dissociated FAS II system that consists of a set of separate enzymes, each catalyzing one of the reactions of the fatty acid synthase cycle (1). A third system exists in some parasites that use membrane-bound fatty acid elongases for the synthesis of aliphatic chains (2). Despite this considerable variation, the individual reaction steps of fatty acid biosynthesis are essentially conserved in all kingdoms of life. Four basic reactions constitute a single round of elongation. In the first step, an acceptor CoA or acyl carrier protein (ACP) associated acetyl unit is condensed with malonyl-ACP to form ␤-ketobutyryl-ACP, which is subsequently reduced by an NADPH-dependent ketoacyl-ACP reductase. The resulting ␤-hydroxyacyl-ACP is dehydrated to produce enoyl-ACP and finally reduced by an enoyl reductase (ER) to form the saturated acyl-ACP, which can be further elongated in a new cycle [see supporting information (SI) Fig. S1].The importance of the fatty acid biosynthesis pathway makes the FAS sys...

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Thiolactomycin, a new antibiotic. I. Taxonomy of the producing organism, fermentation and biological properties.

Cited by 153 publications

References 15 publications

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

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

Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12

Inhibition of the fungal fatty acid synthase type I multienzyme complex

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