RESUMEN
Food allergies are a significant health issue worldwide. In many countries, labeling of primary allergens in food products has been made mandatory to ensure consumer safety. In food manufacturing settings, the lateral flow immunoassay (LFI)-based on antigen-antibody reactions-is a rapid and accurate method for allergen testing and is widely used. Peptide arrays are tools that enable the synthesis of peptides of any sequence on a substrate and high-throughput analysis of their interactions with chemicals. This study aimed to investigate a new application of peptide arrays in the field of food technology, particularly in the development of antibodies for food allergen testing. First, monoclonal antibodies against hen egg ovalbumin, a major food allergen, were produced. Then, using a peptide array, the epitope and specificity of the antibodies were comprehensively and precisely analyzed. Finally, an LFI kit incorporating the antibodies demonstrated both high specificity and detection sensitivity for food allergen testing. These findings indicate that peptide arrays are valuable tools in the development of antibodies for food allergen testing, ensuring reliability and accuracy at the molecular level.
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Retinoid X receptor (RXR), a nuclear receptor (NR) that regulates transcription of target genes in a ligand binding-dependent manner, is of interest as a drug target. RXR agonists have been developed as therapeutic agents for cutaneous invasive T-cell lymphoma (e.g., bexarotene (1)) and investigated as potential anti-inflammatory agents. Screening systems for the binding of RXR alone have been reported. However, although RXRs function as RXR heterodimers, information on systems to evaluate the differential binding of RXR agonists as RXR heterodimers has not been available until recently. Here we show that the fluorescent RXR agonist CU-6PMN (3), designed by our group, can be useful for assessing RXR binding to PPARγ/RXRα, and that the binding data differ from those of RXRα alone. This screening method opens a new avenue for binding assays for RXR heterodimers.
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The flavin-dependent amine oxidase superfamily contains various l-amino acid oxidases (LAAOs) bearing different substrate specificities and enzymatic properties. LAAOs catalyze the oxidation of the α-amino group of l-amino acids (L-AAs) to produce imino acids and H2O2. In this study, an ancestral l-Lys α-oxidase (AncLLysO2) was designed utilizing genome-mined sequences from the Caulobacter species. The AncLLysO2 exhibited high specificity toward l-Lys; the k cat/K m values toward l-Lys were one and two orders larger than those of l-Arg and l-ornithine, respectively. Liquid chromatography-high resolution mass spectrometry analysis indicated that AncLLysO2 released imino acid immediately from the active site after completion of oxidation of the α-amino group. Crystal structures of the ligand-free, l-Lys- and l-Arg-bound forms of AncLLysO2 were determined at 1.4-1.6 Å resolution, indicating that the active site of AncLLysO2 kept an open state during the reaction and more likely to release products. The structures also indicated the substrate recognition mechanism of AncLLysO2; ε-amino, α-amino, and carboxyl groups of l-Lys formed interactions with Q357, A551, and R77, respectively. Biochemical and molecular dynamics simulation analysis of AncLLysO2 indicated that active site residues that indirectly interact with the substrate are also important to exhibit high activity; for example, the aromatic group of Y219 is important to ensure that the l-Lys substrate is placed in the correct position to allow the reaction to proceed efficiently. Taken together, we propose the reaction mechanism of AncLLysO2.
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Oligonucleotide therapeutics have great potential as a next-generation approach to treating intractable diseases. Large quantities of modified DNA/RNA containing xenobiotic nucleic acids (XNAs) must be synthesized before clinical application. In this study, the ancestral RNA ligase AncT4_2 was designed by ancestral sequence reconstruction (ASR) to perform the conjugation reaction of modified RNA fragments. AncT4_2 had superior properties to native RNA ligase 2 from T4 phage (T4Rnl2), including high productivity, a >2.5-fold-higher turnover number, and >10°C higher thermostability. One remarkable point is the broad substrate selectivity of AncT4_2; the activity of AncT4_2 toward 17 of the modified RNA fragments was higher than that of T4Rnl2. The activity was estimated by measuring the conjugation reaction of two RNA strands, 3'-OH (12 bp) and 5'-PO4 (12 bp), in which the terminal and penultimate positions of the 3'-OH fragment and the first and second positions of the 5'-PO4 fragment were substituted by 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-H, respectively. The enzymatic properties of AncT4_2 allowed the enzyme to conjugate large quantities of double-stranded RNA coding for patisiran (>400 µM level), which was formed by four RNA fragments containing 2'-OMe-substituted nucleic acids. Structural analysis of modeled AncT4_2 suggested that protein dynamics were changed by mutation to Gly or indel during ASR and that this may positively impact the conjugation of modified RNA fragments with the enzyme. AncT4_2 is expected to be a key biocatalyst in synthesizing RNA therapeutics by an enzymatic reaction. IMPORTANCE RNA therapeutics is one of the next-generation medicines for treating various diseases. Our designed ancestral RNA ligase AncT4_2 exhibited excellent enzymatic properties, such as high thermal stability, productivity, specific activity, and broad substrate selectivity compared to native enzymes. These advantages create the potential for AncT4_2 to be applied in conjugating the modified RNA fragments containing various xenobiotic nucleic acids. In addition, patisiran, a known polyneuropathy therapeutic, could be synthesized from four fragmented oligonucleotides at a preparative scale. Taken together, these findings indicate AncT4_2 could open the door to synthesizing RNA therapeutics by enzymatic reaction at large-scale production.
Asunto(s)
Ácidos Nucleicos , ARN , ARN/metabolismo , Xenobióticos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , ADN/metabolismo , Ácidos Nucleicos/química , LigasasRESUMEN
Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. In this study, we demonstrate that 3-ethyl-2,5-dimethylpyrazine can be produced from L-Thr by a simple bacterial operon. We conclude that EDMP is synthesized chemoenzymatically from L-Thr via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Aminoacetone is supplied by L-threonine 3-dehydrogenase using L-Thr as a substrate via 2-amino-3-ketobutyrate. Acetaldehyde is supplied by 2-amino-3-ketobutyrate CoA ligase bearing threonine aldolase activity from L-Thr when CoA was at low concentrations. Considering the rate of EDMP production, the reaction intermediate is stable for a certain time, and moderate reaction temperature is important for the synthesis of EDMP. When the precursor was supplied from L-Thr by these enzymes, the yield of EDMP was increased up to 20.2%. Furthermore, we demonstrate that this reaction is useful for synthesizing various alkylpyrazines.
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Retinoid X receptor (RXR) heterodimers such as PPAR/RXR, LXR/RXR, and FXR/RXR can be activated by RXR agonists alone and are therefore designated as permissive. Similarly, existing RXR antagonists show allosteric antagonism toward partner receptor agonists in these permissive RXR heterodimers. Here, we show 1-(3-(2-ethoxyethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazole-5-carboxylic acid (14, CBTF-EE) as the first RXR antagonist that does not show allosteric inhibition in permissive RXR heterodimers. This compound was designed based on the hypothesis that RXR antagonists that do not induce conformational changes of RXR would not exhibit such allosteric inhibition. CD spectra and X-ray co-crystallography of the complex of 14 and the RXR ligand binding domain (LBD) confirmed that 14 does not change the conformation of hRXR-LBD. The X-ray structure analysis revealed that 14 binds at the entrance of the ligand binding pocket (LBP), blocking access to the LBP and thus serving as a "gatekeeper".
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Receptores X Retinoide/antagonistas & inhibidores , Regulación Alostérica , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Ligandos , Receptores X Retinoide/químicaRESUMEN
Exponentially increasing protein sequence data enables artificial enzyme design using sequence-based protein design methods, including full-consensus protein design (FCD). The success of artificial enzyme design is strongly dependent on the nature of the sequences used. Hence, sequences must be selected from databases and curated libraries prepared to enable a successful design by FCD. In this study, we proposed a selection approach regarding several key residues as sequence motifs. We used l-threonine 3-dehydrogenase (TDH) as a model to test the validity of this approach. In the classification, four residues (143, 174, 188, and 214) were used as key residues. We classified thousands of TDH homologous sequences into five groups containing hundreds of sequences. Utilizing sequences in the libraries, we designed five artificial TDHs by FCD. Among the five, we successfully expressed four in soluble form. Biochemical analysis of artificial TDHs indicated that their enzymatic properties vary; half of the maximum measured enzyme activity (t1/2) and activation energies were distributed from 53 to 65 °C and from 38 to 125 kJ/mol, respectively. The artificial TDHs had unique kinetic parameters, distinct from one another. Structural analysis indicates that consensus mutations are mainly introduced in the secondary or outer shell. The functional diversity of the artificial TDHs is due to the accumulation of mutations that affect their physicochemical properties. Taken together, our findings indicate that our proposed approach can help generate artificial enzymes with unique enzymatic properties.
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Oxidorreductasas de Alcohol/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Ingeniería de Proteínas , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Bacterias/química , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Especificidad por Sustrato , TermodinámicaRESUMEN
Ligands for retinoid X receptors (RXRs), "rexinoids", are attracting interest as candidates for therapy of type 2 diabetes and Alzheimer's and Parkinson's diseases. However, current screening methods for rexinoids are slow and require special apparatus or facilities. Here, we created 7-hydroxy-2-oxo-6-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2H-chromene-3-carboxylic acid (10, CU-6PMN) as a new fluorescent RXR agonist and developed a screening system of rexinoids using 10. Compound 10 was designed based on the fact that umbelliferone emits strong fluorescence in a hydrophilic environment, but the fluorescence intensity decreases in hydrophobic environments such as the interior of proteins. The developed assay using 10 enabled screening of rexinoids to be performed easily within a few hours by monitoring changes of fluorescence intensity with widely available fluorescence microplate readers, without the need for processes such as filtration.
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Colorantes Fluorescentes/química , Ligandos , Receptores X Retinoide/agonistas , Umbeliferonas/química , Unión Competitiva , Genes Reporteros , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Unión Proteica , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Tetrahidronaftalenos/química , Umbeliferonas/metabolismoRESUMEN
The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full-consensus design (FCD) and ancestral-sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial l-threonine 3-dehydrogenases (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1, and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH); the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5 °C higher than that of CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were 2- and 7-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.
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Oxidorreductasas de Alcohol/metabolismo , Cupriavidus necator/enzimología , NAD/metabolismo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Biotecnología/métodos , Cristalografía por Rayos X , Cupriavidus necator/química , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Estabilidad de Enzimas , Modelos Moleculares , Filogenia , Plásmidos/genética , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Especificidad por SustratoRESUMEN
A short chain dehydrogenase like l-threonine 3-dehydrogenase (SDR-TDH) from metagenome data (mtTDH) was identified by database mining. Its enzymatic properties suggested that mtTDH has unique characteristics relative to other SDR-TDHs, including two mesophilic and thermophilic SDR-TDHs identified in this study. The activation energy of mtTDH was the lowest (29.6 kJ/mol) of those of the SDR-TDHs, indicating that it is a psychrophilic enzyme. Size-exclusion chromatography analysis revealed mtTDH is a monomer. Crystal structures of mtTDH in apo, binary, and two ternary complexes (l-Ser- and l-Thr-soaked forms) were determined at resolutions of 1.25-1.9 Å. Structural and computational analysis revealed the molecular mechanism of switching between the open and closed states induced by substrate binding and product release. Furthermore, six residues and two water molecules at the active site contributing to product release were assigned. The residues could be categorized into two groups on the basis of the enzymatic properties of their variants: S111, Y136, and T177 and S74, T178, and D179. The former group appeared to affect l-Thr dehydrogenation directly, because the kcat value of their variants was >80-fold lower than that of wild-type mtTDH. On the other hand, the latter group contributes to switching between the open and closed states, which is important for the high substrate specificity of SDR-TDH for l-Thr: the kcat and Km toward l-Thr values of variants in these residues could not be determined because the initial velocity was unsaturated at high concentrations of l-Thr. On the basis of these findings, we proposed a product release mechanism for SDR-TDH associated with specific structural changes.