RESUMEN
Sulforaphane (SFaN) is a food-derived compound with several bioactive properties, including atherosclerosis, diabetes, and obesity treatment. However, the mechanisms by which SFaN exerts its various effects are still unclear. To elucidate the mechanisms of the various effects of SFaN, we explored novel SFaN-binding proteins using SFaN beads and identified acyl protein thioesterase 2 (APT2). We also found that SFaN binds to the APT2 via C56 residue and attenuates the palmitoylation of APT2, thereby reducing plasma membrane localization of APT2. This study reveals a novel bioactivity of SFaN as a regulator of APT2 protein palmitoylation.
Asunto(s)
Isotiocianatos , Lipoilación , Sulfóxidos , Tioléster Hidrolasas , Isotiocianatos/metabolismo , Isotiocianatos/farmacología , Isotiocianatos/química , Sulfóxidos/farmacología , Sulfóxidos/metabolismo , Sulfóxidos/química , Humanos , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/química , Lipoilación/efectos de los fármacos , Unión Proteica , Células HEK293 , Membrana Celular/metabolismoRESUMEN
Macrocyclization of peptides reduces conformational flexibilities, potentially leading to improved drug-like properties, such as target specificities and metabolic stabilities. As chemical methodologies often allow side reactions like epimerization and oligomerization, keen attention has been directed toward enzymatic peptide cyclization using peptide cyclases from specialized metabolic pathways. Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified family of peptide cyclases involved in the biosynthesis of non-ribosomal peptides in actinobacteria. PBP-type TEs have undergone intensive investigation due to their outstanding potential as biocatalysts. This review summarizes the rapidly growing knowledge on PBP-type TEs, with special emphasis on their functions, scopes, and structures, and efforts towards their biocatalytic applications.
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Biocatálisis , Proteínas de Unión a las Penicilinas , Proteínas de Unión a las Penicilinas/metabolismo , Proteínas de Unión a las Penicilinas/química , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , CiclizaciónRESUMEN
Thioesterase (TE) domain exerts a great influence over the structure of the final product and TE-released nonreduced polyketides (nrPKs) retain aromaticity. 3-Methylene isochromanones are lactones with a unique olefin at C3 that disrupts the aromaticity, whose biosynthetic details are speculative. Our study unveils the complete biosynthesis of ascochin, in which the construction of the 3-methylene isochromanone backbone is achieved by a nonreducing polyketide synthase (nrPKS) alone and two subsequent oxidations are involved. Intriguingly, the TEAscD serves as a gatekeeper to direct the product release toward formation of nonaromatic 3-methylene isochromanone, rather than the typical aromatic product.
Asunto(s)
Sintasas Poliquetidas , Estructura Molecular , Oxidación-Reducción , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/química , Policétidos/química , Policétidos/metabolismo , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/química , Palmitoil-CoA Hidrolasa/química , Palmitoil-CoA Hidrolasa/metabolismoRESUMEN
DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.
Asunto(s)
Antituberculosos , Inhibidores Enzimáticos , Mycobacterium tuberculosis , Sintasas Poliquetidas , Bibliotecas de Moléculas Pequeñas , Tioléster Hidrolasas , Animales , Humanos , Ratones , Antituberculosos/química , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon-bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, an unusual "gene-within-gene" thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/ß-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an intracellular tagged carrier-protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.
Asunto(s)
Lípidos , Tioléster Hidrolasas , Dimerización , Tioléster Hidrolasas/química , Mutagénesis Sitio-DirigidaRESUMEN
Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles. In this feature article, we describe our work to characterize the substrate selectivity of TEs and to use these enzymes as biocatalysts. Our efforts have shown that the linear thioester activated substrates are loaded on TEs with limited substrate selectivity to generate acyl-enzyme intermediates. We show that cyclization of the acyl-enzyme intermediates can be highly selective, with competing hydrolysis of the acyl-enzyme intermediates. The mechanisms controlling TE-mediated macrocyclization versus hydrolysis are a significant unsolved problem in TE biochemistry. The potential of TEs as biocatalysts was demonstrated by using them in the chemoenzymatic total synthesis of macrocyclic depsipeptide natural products. This article highlights the strengths and potential of TEs as biocatalysts as well as their limitations, opening exciting research opportunities including TE engineering to optimize these powerful biocatalysts.
Asunto(s)
Tioléster Hidrolasas , Hidrólisis , Tioléster Hidrolasas/químicaRESUMEN
Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.
Asunto(s)
Dominio Catalítico , Unión Proteica , Pliegue de Proteína , Tioléster Hidrolasas , Humanos , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/genética , Ligandos , Modelos Moleculares , Secuencia de Aminoácidos , Cinética , Secuencia Conservada , Estabilidad de Enzimas , Francisella tularensis/enzimología , Francisella tularensis/metabolismo , Francisella tularensis/química , Cristalografía por Rayos X , Especificidad por SustratoRESUMEN
Ubiquitin specific protease 30 (USP30) has been attributed to mitochondrial dysfunction and impediment of mitophagy in Parkinson's disease (PD). This happens once ubiquitin that supposed to bind with deformed mitochondria at the insistence of Parkin, it's been recruited by USP30 via the distal ubiquitin binding domain. This is a challenge when PINK1 and Parkin loss their functions due to mutation. Although, there are reports on USP30s' inhibitors but no study on the repurposing of inhibitors approved against MMP-9 and SGLT-2 as potential inhibitors of USP30 in PD. Thus, the highlight therein, is to repurpose approved inhibitors of MMP-9 and SGLT-2 against USP30 in PD using extensive computational modelling framework. 3D structures of Ligands and USP30 were obtained from PubChem and protein database (PDB) servers respectively, and were subjected to molecular docking, ADMET evaluation, DFT calculation, molecular dynamics simulation (MDS) and free energy calculations. Out of the 18 drugs, 2 drugs showed good binding affinity to the distal ubiquitin binding domain, moderate pharmacokinetic properties and good stability. The findings showed canagliflozin and empagliflozin as potential inhibitors of USP30. Thus, we present these drugs as repurposing candidates for the treatment of PD. However, the findings in this current study needs to be validated experimentally.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Metaloproteinasa 9 de la Matriz , Simulación del Acoplamiento Molecular , Reposicionamiento de Medicamentos , Proteínas Quinasas/metabolismo , Proteínas Mitocondriales/química , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Characterization of thioesterases (TEs) is an important step in understanding natural product biosynthesis. Studying non-ribosomal peptide synthetase (NRPS) TEs presents a unique set of challenges with specific cloning and expression issues as well as the challenging synthesis of the thioester peptides substrate required for characterization of the TE. In this method, we describe the cloning and expression of NRPS TEs, the synthesis of thioester peptides, and the in vitro biochemical characterization of the enzyme.
Asunto(s)
Péptidos , Tioléster Hidrolasas , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismo , Péptidos/química , Péptido Sintasas/químicaRESUMEN
Methylglyoxal (MG) is a reactive and toxic compound produced in carbohydrate, lipid, and amino acid metabolism. The glyoxalase system is the main detoxifying route for MG and consists of two enzymes, glyoxalase I (GlxI) and glyoxalase II (GlxII). GlxI catalyzes the formation of S-d-lactoylglutathione from hemithioacetal, and GlxII converts this intermediate to d-lactate. A relationship between the glyoxalase system and some diseases like diabetes has been shown, and inhibiting enzymes of this system may be an effective means of controlling certain diseases. A detailed understanding of the reaction mechanism of an enzyme is essential to the rational design of competitive inhibitors. In this work, we use quantum mechanics/molecular mechanics (QM/MM) calculations and energy refinement utilizing the big-QM and QM/MM thermodynamic cycle perturbation methods to propose a mechanism for the GlxII reaction that starts with a nucleophilic attack of the bridging OH- group on the substrate. The coordination of the substrate to the Zn ions places its electrophilic center close to the hydroxide group, enabling the reaction to proceed. Our estimated reaction energies are in excellent agreement with experimental data, thus demonstrating the reliability of our approach and the proposed mechanism. Additionally, we examined alternative protonation states of Asp-29, Asp-58, Asp-134, and the bridging hydroxide ion in the catalytic process. However, these give less favorable reactions, a poorer reproduction of the crystal structure geometry of the active site, and higher root-mean-squared deviations of the active site residues in molecular dynamics simulations.
Asunto(s)
Simulación de Dinámica Molecular , Tioléster Hidrolasas , Lactoilglutatión Liasa/química , Lactoilglutatión Liasa/metabolismo , Reproducibilidad de los Resultados , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismoRESUMEN
The Neuronal Ceroid Lipofuscinoses (NCL) are a group of progressive neurodegenerative disorders, associated with 14 Ceroid Lipofuscinosis Neuronal genes (CLN1-14). The mutations in the Palmitoyl-Protein Thioesterase 1 (PPT1) protein serve as one of the major reasons for the causative of NCL. The PPT1 involves degrading and modifying cysteine residues in proteins or peptides by removing thioester-linked fatty acyl groups like palmitate prefers acyl chains of 14-18 carbons in length. In this study, we have analyzed the impact of PPT1 mutations on the deleteriousness, stability, conservative nature of amino acid, and impact of mutations on the protein structure. We have also used molecular dynamics simulations using GROMACS to perceive the alteration in the dynamic behavior of the PPT1 at the residual level. In this study, we have retrieved 23 PPT1 mutations from the UniProt database, and these were subjected to a series of analyses using varied computer algorithms. From these analyses, out of 23 mutations, 16 mutations were identified as deleterious. Among 16, eight mutations were identified to destabilize the protein structure, and finally, two mutations (W38C and L222P) were found to be positioned in the highly conserved region. The structural impact study observed that the mutant proline could disrupt the alpha helix formed by the leucine at position 222. Finally, from the molecular dynamics simulations, we observed that due to the mutations (W38C and L222P), the protein had experienced higher deviation, fluctuation, and lower compactness. These structural changes elucidate that these mutations can impact the structure and function of the PPT1 protein.
Asunto(s)
Lipofuscinosis Ceroideas Neuronales , Tioléster Hidrolasas/metabolismo , Humanos , Proteínas de la Membrana/genética , Mutación , Lipofuscinosis Ceroideas Neuronales/genética , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genéticaRESUMEN
Thioesterases play a critical role in metabolism, membrane biosynthesis, and overall homeostasis for all domains of life. In this present study, we characterize a putative thioesterase from Escherichia coli MG1655 and define its role as a cytosolic enzyme. Building on structure-guided functional predictions, we show that YigI is a medium- to long-chain acyl-CoA thioesterase that is involved in the degradation of conjugated linoleic acid (CLA) in vivo, showing overlapping specificity with two previously defined E. coli thioesterases TesB and FadM. We then bioinformatically identify the regulatory relationships that induce YigI expression, which include: an acidic environment, high oxygen availability, and exposure to aminoglycosides. Our findings define a role for YigI and shed light on why the E. coli genome harbors numerous thioesterases with closely related functions. IMPORTANCE Previous research has shown that long chain acyl-CoA thioesterases are needed for E. coli to grow in the presence of carbon sources such as conjugated linoleic acid, but that E. coli must possess at least one such enzyme that had not previously been characterized. Building off structure-guided function predictions, we showed that the poorly annotated protein YigI is indeed the previously unidentified third acyl CoA thioesterase. We found that the three potentially overlapping acyl-CoA thioesterases appear to be induced by nonoverlapping conditions and use that information as a starting point for identifying the precise reactions catalyzed by each such thioesterase, which is an important prerequisite for their industrial application and for more accurate metabolic modeling of E. coli.
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Escherichia coli , Ácidos Grasos , Tioléster Hidrolasas/metabolismo , Acilcoenzima A/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genéticaRESUMEN
Oxidative coupling and oxidative rearrangement are two of the most common biosynthetic strategies to form diaryl ethers. In contrast, enzymatic diaryl ether generation that proceeds in a nonoxidative manner has not been characterized thus far. Here, we discovered a versatile thioesterase (TE) domain from the nonreducing polyketide synthase (nrPKS) AN7909, which catalyzes diaryl ether formation through a series of successive steps involving esterification, a Smiles rearrangement, and hydrolysis. Further mutations and biochemical analyses with synthetic mimic substrates provide insight into the proposed catalytic process of the TE domain.
Asunto(s)
Éter , Sintasas Poliquetidas , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Tioléster Hidrolasas/químicaRESUMEN
Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.
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Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , Péptido Sintasas/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismo , Catálisis , Escherichia coli/genética , Ácido Graso Sintasas/genética , Ingeniería Metabólica , Dominios Proteicos , Tioléster Hidrolasas/genéticaRESUMEN
Thioesterases are enzymes that hydrolyze thioester bonds in numerous biochemical pathways, for example in fatty acid synthesis. This work reports known functions, structures, and mechanisms of updated thioesterase enzyme families, which are classified into 35 families based on sequence similarity. Each thioesterase family is based on at least one experimentally characterized enzyme, and most families have enzymes that have been crystallized and their tertiary structure resolved. Classifying thioesterases into families allows to predict tertiary structures and infer catalytic residues and mechanisms of all sequences in a family, which is particularly useful because the majority of known protein sequence have no experimental characterization. Phylogenetic analysis of experimentally characterized thioesterases that have structures with the two main structural folds reveal convergent and divergent evolution. Based on tertiary structure superimposition, catalytic residues are predicted.
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Tioléster Hidrolasas , Secuencia de Aminoácidos , Catálisis , Humanos , Filogenia , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.
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Bacillus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Tioléster Hidrolasas/química , beta-Lactamasas/química , Ampicilina/química , Ampicilina/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular , Glutatión/análogos & derivados , Glutatión/química , Glutatión/metabolismo , Simulación del Acoplamiento Molecular , Filogenia , Conformación Proteica , Stenotrophomonas maltophilia/enzimologíaRESUMEN
ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed 'DarT' from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , ADN/metabolismo , Tioléster Hidrolasas/fisiología , Adenosina Difosfato Ribosa/química , Toxinas Bacterianas/metabolismo , Línea Celular , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple/metabolismo , Humanos , Tioléster Hidrolasas/química , Timidina/metabolismoRESUMEN
The standalone metallo-ß-lactamase-type thioesterase (MßL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MßL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MßL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two ß strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance.
Asunto(s)
Sitios de Unión/genética , Mutagénesis Sitio-Dirigida/métodos , Tioléster Hidrolasas , beta-Lactamasas , Antracenos/metabolismo , Estabilidad de Enzimas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Eurotiales/enzimología , Eurotiales/genética , Proteínas Fúngicas/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Perfluorooctanoic acid (PFOA), a typical representative of per- and polyfluoroalkyl substances (PFASs), is a widely utilized persistent organic pollutant (POP) known to induce liver toxicity in laboratory animals and wildlife. Evidence suggests that PFOA interacts with Acyl-CoA thioesterase 1 (Acot1) to modulate levels of ß-oxidation. Specifically, PFOA accelerates ß-oxidation, while Acot1 is inhibitory. Few studies have investigated the specific relationship between PFOA and Acot1 and the mechanism of their interaction remains unclear. In the following study, purified rat Acot1 protein was synthesized via bacterial recombination and the structural features that facilitate its binding to PFOA were assessed via molecular docking technology. Additionally, through use of circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC) we demonstrate that PFOA binds to WT-Acot1 through electrostatic attraction and low strength non-covalent hydrogen bonding at a molar ratio of 1:1. Furthermore, we identify N326 and H373 amino acid residues as key regulators of the binding process. Together, these findings clarify the interaction pattern of PFOA and Acot1 proteins and provide insight into the specific molecular mechanisms that induce PFOA toxicity in humans and animals.
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Caprilatos/química , Fluorocarburos/química , Hígado/enzimología , Tioléster Hidrolasas/química , Animales , Caprilatos/metabolismo , Fluorocarburos/metabolismo , Hígado/metabolismo , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Proteolisis , Ratas , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Nature has always been seemingly limitless in its ability to create new chemical entities. It provides vastly diverse natural compounds through a biomanufacturing process that involves myriads of biosynthetic machineries. Here we report a case of unusual formations of hybrid natural products that are derived from two distinct polyketide biosynthetic pathways, the NFAT-133 and conglobatin pathways, in Streptomyces pactum ATCC 27456. Their chemical structures were determined by NMR spectroscopy, mass spectrometry, and chemical synthesis. Genome sequence analysis and gene inactivation experiments uncovered the biosynthetic gene cluster of conglobatin in S. pactum. Biochemical studies of the recombinant thioesterase (TE) domain of the conglobatin polyketide synthase (PKS) as well as its S74A mutant revealed that the formation of these hybrid compounds requires an active TE domain. We propose that NFAT-133 can interfere with conglobatin biosynthesis by reacting with the TE-domain-bound intermediates in the conglobatin PKS assembly line to form hybrid NFAT-133/conglobatin products.