Abstract:YbgC proteins are bacterial acyl-CoA thioesterases associated with the Tol-Pal system. This system is important for cell envelope integrity and is part of the cell division machinery. In E. coli, YbgC associates with the cell membrane and is part of a protein network involved in lipid biogenesis. In the human pathogen Helicobacter pylori, a putative homologue of YbgC, named HP0496, was found to interact with the cytotoxin CagA by two different studies. We have determined its crystal structure and characterized… Show more
“…This buried dimer interface (4540 Å 2 ) is stabilized via main-chain interactions between strands 2 and by residues 25-34 of the central helix ␣1 with their 2-fold counterparts, as well as by hydrophobic interactions between ␣2 of one subunit and the 1-␣1 loop of the other subunit. Tetramer formation leads to the burying of the four central helices while the -sheets are exposed to the solvent, as observed in other hotdog fold thioesterases with the ⑀␥ oligomeric arrangement (24,29,30). A hydrophilic channel ϳ8 Å wide lies at the center of the tetramer along a 2-fold symmetry axis.…”
Section: Resultsmentioning
confidence: 86%
“…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.…”
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...
“…This buried dimer interface (4540 Å 2 ) is stabilized via main-chain interactions between strands 2 and by residues 25-34 of the central helix ␣1 with their 2-fold counterparts, as well as by hydrophobic interactions between ␣2 of one subunit and the 1-␣1 loop of the other subunit. Tetramer formation leads to the burying of the four central helices while the -sheets are exposed to the solvent, as observed in other hotdog fold thioesterases with the ⑀␥ oligomeric arrangement (24,29,30). A hydrophilic channel ϳ8 Å wide lies at the center of the tetramer along a 2-fold symmetry axis.…”
Section: Resultsmentioning
confidence: 86%
“…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.…”
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...
“…YbgC proteins, found only in bacteria, belong to the hot-dog thioesterase superfamily 16 . Consistently, esterase/thioesterase activity (acyl-CoA hydrolase) for YbgC proteins in E. coli , Haemophilus influenzae , and Helicobacter pylori has been demonstrated; as a consequence, it has been proposed that YbgC proteins might be involved in the biosynthesis of species-specific phospholipids 17–19 .Figure 1Organization of the tol-pal cluster in representative bacteria. Genes flanking the tol - pal cluster vary.…”
Section: Introductionmentioning
confidence: 72%
“…S. oneidensis YbgC is annotated to be a thioesterase and its counterparts in bacteria, such as E. coli , H. influenzae , and H. pylori , show thioesterase activity although their substrates differ 17, 19, 52 . A sequence analysis revealed a comfortable sequence similarity (against Ec YbgC, E-value, 5e-40) between S. oneidensis YbgC and those whose thioesterase activity had been established (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The thioesterase activity of YbgC was determined by the difference in UV-visible light absorption between the substrate and the hydrolytic product as described previously 19 . In brief, hydrolysis reactions of the aryl-CoA substrates (Sigma-Aldrich) were monitored at 25 °C by recording the absorbance of 5-thio-2-nitrobenzoate at 412 nm, which was formed by 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) with CoASH released after acyl-CoA hydrolysis for 20 min.…”
Because of ubiquity of thioesters, thioesterases play a critical role in metabolism, membrane biosynthesis, signal transduction, and gene regulation. In many bacteria, YbgC is such an enzyme, whose coding gene mostly resides in the tol-pal cluster. Although all other proteins encoded in the tol-pal cluster are clearly involved in maintaining cell envelope integrity and cell division, little is known about the physiological role of YbgC. In this study, we identify in Shewanella oneidensis, a γ-proteobacterium used as a research model for environmental microbes, YbgC as a motility regulator. The loss of YbgC results in enhanced motility, which is likely due to the increased rotation rate of the flagellum. The regulatory function of YbgC requires its thioesterase activity but could not be replaced by YbgC homologues of other bacteria. We further show that the regulation of YbgC is mediated by the second message c-di-GMP.
A series of new hypervalent iodine reagents based on the 1,3-dihydro-3,3-dimethyl-1,2-benziodoxole and 1,2-benziodoxol-3-(1H)-one scaffolds, which contain a functionalized tetrafluoroethyl group, have been prepared, characterized, and used in synthetic applications. Their corresponding electrophilic fluoroalkylation reactions with various sulfur, oxygen, phosphorus, and carbon-centered nucleophiles afford products that feature a tetrafluoroethylene unit, which connects two functional moieties. A related λ(3) -iodane that contains a fluorophore was shown to react with a cysteine derivative under mild conditions to give a thiol-tagged product that is stable in the presence of excess thiol. Therefore, these new reagents show a significant potential for applications in chemical biology as tools for fast, irreversible, and selective thiol bioconjugation.
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