Identification of a Type III Thioesterase Reveals the Function of an Operon Crucial for Mtb Virulence

Wang, Feng; Langley, Robert; Gulten, Gulcin; Wang, Lei; Sacchettini, James C.

doi:10.1016/j.chembiol.2007.04.005

Cited by 45 publications

(56 citation statements)

References 33 publications

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“…In the course of analyzing the M. tuberculosis genome for FabZ homologues, Rv0098, a long-chain fatty acyl-CoA thioesterase (FcoT) with only low catalytic activity (42), was revealed as a promising candidate (38,41). Rv0098 is 36% identical and 54% similar to Streptococcus pneumoniae FabZ and contains the latter's conserved active-site residues that are also present in E. coli FabZ and FabA (41).…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

2009

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We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2⌬ cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2⌬ mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2⌬ cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2⌬ cells also restored de novo lipoic acid synthesis to 92 and 40% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.Mycobacterium tuberculosis causes immense human morbidity and mortality worldwide, and it is thought that about 30 million people have died from tuberculosis in the past decade alone (see references 1 and 41 and citations therein). The World Health Organization estimates that one-third of the human population is infected with M. tuberculosis, which kills more adults than any other single infectious agent (43). Although effective drugs against tuberculosis exist, treatment is extended and arduous, and in certain countries 36% of tuberculosis patients are now infected with isoniazid-or rifampinresistant strains (36). Hence, there is a renewed interest and urgency in developing new therapeutics against M. tuberculosis.An attractive target for therapeutics is represented by the essential process of bacterial fatty acid biosynthesis (10, 24). M. tuberculosis contains a type 2 fatty acid synthase system (FASII) that consists of discrete enzymes, but it also has an additional associative FASI system (5) which is comprised of several enzymatic activities within a single multifunctional synthase. The two FAS systems cooperate in the production of mycolic acids, ...

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Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

2009

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“…rv0097 is part of an operon, rv0096-rv0101 (16), that is required for survival of M. tuberculosis in mouse macrophages (17). Recently, rv0098 was identified as a type III thioesterase, and it was hypothesized that this enzyme hydrolyzes fatty acyl-CoA substrates to generate free C 16 -C 18 fatty acids, which are used in the synthesis of lipopeptides (18). Other genes within the operon encode a PPE protein family member (Rv0096), a fatty acid AMP ligase (Rv0099), an acyl carrier protein (Rv0100), and a nonribosomal peptide synthase (Rv0101).…”

Section: Tuberculosis Persistence Mutantsmentioning

confidence: 99%

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

Dhar

McKinney

2010

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

113

106

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Tuberculosis (TB) is notoriously difficult to cure, requiring administration of multiple antibiotics for 6 mo or longer. Conventional anti-TB drugs inhibit biosynthetic processes involved in cell growth and division, such as DNA replication, RNA transcription, protein translation, and cell wall biogenesis. Although highly effective against bacteria cultured in vitro under optimal growth conditions, these antibiotics are less effective against bacteria grown in vivo in the tissues of a mammalian host. The factors that contribute to the antibiotic tolerance of bacteria grown in vivo are unknown, although altered metabolism and sluggish growth are hypothesized to play a role. To address this question, we identified mutations in Mycobacterium tuberculosis that impaired or enhanced persistence in mice treated with isoniazid (INH), a front-line anti-TB drug. Disruption of cydC, encoding a putative ATP-binding cassette transporter subunit, accelerated bacterial clearance in INH-treated mice without affecting growth or survival in untreated mice. Conversely, transposon insertions within the rv0096-rv0101 gene cluster attenuated bacterial growth and survival in untreated mice but paradoxically prevented INH-mediated killing of bacteria in treated mice. These contrasting phenotypes were dependent on the interaction of the bacteria with the tissue environment because both mutants responded normally to INH when grown in macrophages ex vivo or in axenic cultures in vitro. Our findings have important implications because persistence-impairing mutations would be missed by conventional genetic screens to identify candidate drug targets. Conversely, persistenceenhancing mutations would be missed by standard diagnostic methods, which are performed on bacteria grown in vitro, to detect drug resistance.chemotherapy | drug tolerance | host environment T uberculosis (TB) has been a treatable disease for more than half a century, yet TB eradication remains a distant and possibly unachievable goal barring the introduction of more effective control strategies (1). A formidable obstacle to successful treatment of TB is the requirement for frequent administration of multiple drugs for 6 mo or longer (2). Unless drug administration is closely supervised, the majority of patients will not adhere to such a protracted therapeutical regimen (2). Consequently, patient nonadherence is responsible for high rates of treatment failure, relapse, and emergent drug resistance (3). To reduce treatment costs and improve patient adherence, there is an urgent need for more effective anti-TB drugs to shorten the treatment time (2, 4).Conventional anti-TB drugs target biosynthetic processes involved in cell growth, including RNA transcription [rifampicin (RIF)], protein translation (streptomycin), and cell wall biogenesis [isoniazid (INH)]. Although these drugs rapidly kill Mycobacterium tuberculosis grown in vitro, they are less active against bacteria grown in vivo in mammalian hosts (4,5). In the mouse model of TB, infection progresses through a brief ac...

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“…Such hotdog fold proteins include many characterized and hypothetical thioesterases that use acyl CoA as substrates (23). The three-dimensional structure and substrate specificity of several hotdog fold thioesterases have been determined, including YbgC from Helicobacter pylori (24), Paal from E. coli (25), HB8 from Thermos thermophilis (26), FcoT from Mycobacterium tuberculosis (27), YciA from Haemophilus influenzae (28), human THEM2 (25) and 4-hydroxylbenzoyl-CoA thioesterases (4-HBT) from Pseudomonas sp. Strain CBS and Arthrobacter sp.…”

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confidence: 99%

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis

Kotaka¹,

Kong²,

Qureshi

et al. 2009

Journal of Biological Chemistry

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The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg 37 plays a crucial role in the hydrolysis of the thioester bond, and that Tyr 29 and a hydrogen-bonded water network assist the decarboxylation of the ␤-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4‫؍‬C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.Enediyne natural products represent a family of structurally unique secondary metabolites with potent antitumor and antibiotic activities. Based on the structure of the bicyclic enediyne core, enediyne natural products are categorized into two groups with either a 9-or 10-membered enediyne moiety (1, 2). The antitumor activity of enediyne natural products derives from their capacity to induce chromosomal DNA cleavage through an oxidative radical mechanism (3). The biosynthetic mechanism for the enediyne moiety has been, however, elusive despite clues gleaned from early isotope-feeding experiments (4, 5). Pioneering genetic studies of the biosynthesis of calicheamicin and C-1027 from two research groups yielded major insights into the biosynthetic pathways, suggesting that an iterative polyketide synthase (PKS) 5 plays a central role in the assembly of both the 9-and 10-membered enediyne moieties (6, 7). The gene clusters also contain open reading frames encoding hypothetical proteins for the downstream processing of the PKS product. The involvement of similar genes in enediyne biosynthesis was later confirmed for neocarzinostatin, maduropeptin, dynemicin, and several putative enediyne natural products in soil and marine microorganisms (8 -11). Recently, based on the study on the 9-membered enediynecontaining C-1027, Shen and coworkers found that the iterative PKS (SgcE) and the putative thioesterase (SgcE10) generated a conjugated polyene (1,3,5,7,9,11,13-pentadecaheptaene) through an ACP-tethered 3-hydroxy-4...

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Identification of a Type III Thioesterase Reveals the Function of an Operon Crucial for Mtb Virulence

Cited by 45 publications

References 33 publications

Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis

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