RESUMO
D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.
Assuntos
Aminobutiratos , Caenorhabditis elegans , D-Aminoácido Oxidase , Escherichia coli , Engenharia de Proteínas , D-Aminoácido Oxidase/metabolismo , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Engenharia de Proteínas/métodos , Animais , Aminobutiratos/metabolismo , Aminobutiratos/química , Desaminação , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/químicaRESUMO
This study focuses on the purification and detection of glufosinate (GLUF) and its metabolites N-acetyl GLUF and MPP in plasma samples. A Dikma Polyamino HILIC column was used for the effective retention and separation of GLUF and its metabolites, and the innovative addition of a low concentration of ammonium fluoride solution to the mobile phase effectively improved the detection sensitivity of the target analytes. Monodisperse core-shell weak cation exchange (WCX)/C18 bifunctional magnetic polymer composites (Fe3O4@WCX/C18) were prepared in a controllable manner, and their morphology and composition were fully characterized. The Fe3O4@WCX/C18 microspheres were used as a magnetic solid-phase extraction (MSPE) adsorbent for the sample purification and detection of GLUF and its metabolites in plasma samples combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The purification conditions of Fe3O4@WCX/C18 microspheres for GLUF and its metabolites in spiked plasma samples were optimized to achieve the best MSPE efficiency. The purification mechanisms of the target analytes in plasma samples include electrostatic attraction and hydrophobic interactions. Furthermore, the effect of the molar ratio of the two functional monomers 4-VBA and 1-octadecene in the adsorbent was optimized and it shows that the bifunctional components WCX/C18 have a synergistic effect on the determination of GLUF and its metabolites in plasma samples. In addition, the present study compared the purification performance of the Fe3O4@WCX/C18 microsphere-based MSPE method with that of the commercial Oasis WCX SPE method, and the results showed that the Fe3O4@WCX/C18 microsphere-based MSPE method established in this work had a stronger ability to remove matrix interferences. Under optimal purification conditions, the recoveries of GLUF and its metabolites in plasma were 87.6-111 % with relative standard deviations (RSDs) ranging from 0.2 % to 4.8 %. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.10-0.18 µg/L and 0.30-0.54 µg/L, respectively. The MSPE-LC-MS/MS method developed in this study is fast, simple, accurate and sensitive and can be used to confirm GLUF intoxication based not only on the detection of the GLUF prototype but also on the detection of its two metabolites.
Assuntos
Aminobutiratos , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Aminobutiratos/sangue , Aminobutiratos/química , Cromatografia Líquida/métodos , Limite de Detecção , Polímeros/química , Animais , Microesferas , Adsorção , Ratos , Cromatografia por Troca Iônica/métodosRESUMO
Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and α-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus. Bioinformatic analysis revealed that several Streptomyces viridifaciens and Kitasatospora aureofaciens strains also have this cluster. One S. viridifaciens strain was confirmed to produce cirratiomycin. The biosynthetic gene cluster was shown to be responsible for cirratiomycin biosynthesis in S. cirratus in a gene inactivation experiment using CRISPR-cBEST. Interestingly, this cluster encodes a nonribosomal peptide synthetase (NRPS) composed of 12 proteins, including those with an unusual domain organization: a stand-alone adenylation domain, two stand-alone condensation domains, two type II thioesterases, and two NRPS modules that have no adenylation domain. Using heterologous expression and inâ vitro analysis of recombinant enzymes, we revealed the biosynthetic pathway of (2S,3S)-DABA: (2S,3S)-DABA is synthesized from l-threonine by four enzymes, CirR, CirS, CirQ, and CirB. In addition, CirH, a glycine/serine hydroxymethyltransferase homolog, was shown to synthesize α-(hydroxymethyl)serine from d-serine inâ vitro. These findings broaden our knowledge of nonproteinogenic amino acid biosynthesis.
Assuntos
Vias Biossintéticas , Família Multigênica , Serina , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Serina/química , Serina/biossíntese , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Aminobutiratos/química , Aminobutiratos/metabolismo , Antibacterianos/biossíntese , Antibacterianos/químicaRESUMO
We have used a variety of theoretical and experimental techniques to study the role of four basic amino acids-Arginine, Lysine, Ornithine and L-2,4-Diaminobutyric acid-on the structure, flexibility and sequence-dependent stability of DNA. We found that the presence of organic ions stabilizes the duplexes and significantly reduces the difference in stability between AT- and GC-rich duplexes with respect to the control conditions. This suggests that these amino acids, ingredients of the primordial soup during abiogenesis, could have helped to equalize the stability of AT- and GC-rich DNA oligomers, facilitating a general non-catalysed self-replication of DNA. Experiments and simulations demonstrate that organic ions have an effect that goes beyond the general electrostatic screening, involving specific interactions along the grooves of the double helix. We conclude that organic ions, largely ignored in the DNA world, should be reconsidered as crucial structural elements far from mimics of small inorganic cations.
Assuntos
Aminoácidos Básicos , Sequência de Bases , DNA , Aminoácidos Básicos/análise , Aminoácidos Básicos/química , Aminobutiratos/química , Composição de Bases , DNA/análise , DNA/química , Simulação de Dinâmica Molecular , Origem da Vida , TermodinâmicaRESUMO
According to current regulatory guidelines, a stability-indicating method has been developed to determine the impurities in sacubitril (SCB) and valsartan (VLS) tablet dosage forms and perform robustness studies using the design of experiments approach. The present study was initiated to understand quality target product profile, analytical target profile, and risk assessment for method variables that affect the method response. A reversed-phase-HPLC system was equipped with a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 µm) and a photo diode array detector. A gradient mobile phase was used in this research work. The detection was performed at 254 nm; the flow rate was 1.5 mL/min, and the column temperature was maintained at 30°C. The proposed method was validated per the International Council for Harmonisation Q2 (R1) guidelines. The coefficient of correlation was >0.999 for all impurities. The limits of detection and quantification were evaluated for SCB, VLS, and all impurities. The precision and accuracy were obtained for SCB, VLS, and their related impurities. Intra- and inter-day relative standard deviation values were less than 10.0%, and the recoveries of impurities varied between 90.0 and 115.0%. Based on the validation results, the proposed DoE method can estimate SCB and VLS impurities in the finished dosage form.
Assuntos
Aminobutiratos , Compostos de Bifenilo , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Medicamentos , Valsartana , Aminobutiratos/análise , Aminobutiratos/química , Compostos de Bifenilo/análise , Compostos de Bifenilo/química , Cromatografia de Fase Reversa , Combinação de Medicamentos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Projetos de Pesquisa , Valsartana/análise , Valsartana/químicaRESUMO
Chitooligosaccharide grafted with ß-aminobutyric acid based on the idea of bioactive molecular splicing was prepared, and the differences in drought resistance activity before and after grafting were compared. The mechanism was investigated by comparing the differences of the derivative with the Control and Drought about metabolomes. The results showed that the expected derivative was successfully synthesized, named COS-BABA, and had better drought resistance-inducing activity than the raw materials. We suggest that COS-BABA induced drought resistance through second messenger-induced activation of signaling pathways related to traumatic acid and indol-3-lactic acid, which enhanced nucleic acid metabolism to accumulate nucleotides and decreased some amino acids to facilitate protein synthesis. These proteins are regulated to strengthen photosynthesis, resulting in the promotion of carbohydrate metabolism. The accumulation of unsaturated fatty acids stabilized the cell membrane structure and prevented nonstomatal water dissipation. This study provides ideas for the development of more effective drought resistance inducers.
Assuntos
Aminobutiratos/química , Quitosana/química , Secas , Metabolômica/métodos , Oligossacarídeos/química , Plântula/química , Triticum/química , Aminobutiratos/metabolismo , Metabolismo dos Carboidratos , Quitosana/metabolismo , Peso Molecular , Ácidos Nucleicos/metabolismo , Oligossacarídeos/metabolismo , Fotossíntese , Folhas de Planta/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Estresse Fisiológico , Água/químicaRESUMO
Condensation (C) domains in non-ribosomal peptide synthetases catalyze peptide elongation steps whereby activated amino acid or peptidyl acyl donors are coupled with specific amino acid acceptors. In the biosynthesis of the ß-lactam antibiotic nocardicin A, an unusual C domain converts a seryl tetrapeptide into its pentapeptide product containing an integrated ß-lactam ring. While indirect evidence for the intermediacy of a dehydroalanyl species has been reported, here we describe observation of the elusive enzyme-bound dehydroamino acyl intermediate generated from the corresponding allo-threonyl tetrapeptide and partitioned into pentapeptide products containing either a dehydrobutyrine residue or an embedded ß-lactam. Contrary to trends in the literature where condensation domains have been deemed flexible as to acyl donor structure, this ß-lactam synthesizing domain is highly discriminating. The observation of dehydrobutyrine formation links this C domain to related clades associated with natural products containing dehydroamino acid and d-configured residues, suggesting a common mechanistic link.
Assuntos
Antibacterianos/química , Lactamas/química , Oligopeptídeos/química , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminobutiratos/química , Antibacterianos/farmacologia , Catálise , Liberação Controlada de Fármacos , Lactamas/farmacologia , Relação Estrutura-AtividadeRESUMO
Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.
Assuntos
Fármacos Antiobesidade/química , Obesidade/tratamento farmacológico , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Tecido Adiposo/efeitos dos fármacos , Alcinos/química , Aminobutiratos/química , Animais , Fármacos Antiobesidade/farmacologia , Apetite/efeitos dos fármacos , Ácido Aspártico/química , Modelos Animais de Doenças , Descoberta de Drogas , Ácido Glutâmico/química , Glicina/análogos & derivados , Glicina/química , Células HEK293 , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Insulina/metabolismo , Lactamas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Triazóis/químicaRESUMO
In order to better explain and predict the dissolution characteristics of binary drug delivery systems (BDDSs), the dissolution behaviors of co-crystal (CC) and co-amorphous (CA) systems of sacubitril (SCB) and valsartan (VST) were evaluated in vitro and in vivo by thermodynamic and kinetic methods. The CCs of SCB and VST were prepared into a CA state through rotary evaporation. Solid-state properties were systematically evaluated. Herein, based on the results from previous studies of single-phase systems, we used thermodynamic methods to evaluate the increase in drug dissolution rate after BDDSs change from the crystalline to the amorphous state. After comparing the predicted and measured dissolution rate enhancement of the CC and CA systems, this paper attempts to explain the dissolution rate characteristics of the BDDSs. We then evaluated the bioavailability of two BDDSs in beagle dogs to confirm that there was no discrepancy in vivo with the results obtained in vitro. The results exhibited that there is strong intermolecular interaction between SCB and VST and good physical stability for the CA system. Compared with the CC, the bioavailability of SCB and VST in the CA system increased by 313.9% and 130.5%, respectively. The predicted dissolution rate ratio between CC and CA systems and their actual intrinsic dissolution rates differed by only a factor of 2.5, demonstrating the good correlation between the predicted and measured values. In the future, this method could be expanded to a variety of new samples and exciting drug prospects.
Assuntos
Aminobutiratos/administração & dosagem , Antagonistas de Receptores de Angiotensina/administração & dosagem , Anti-Hipertensivos/administração & dosagem , Sistemas de Liberação de Medicamentos , Tetrazóis/administração & dosagem , Termodinâmica , Valsartana/administração & dosagem , Aminobutiratos/química , Aminobutiratos/farmacocinética , Antagonistas de Receptores de Angiotensina/química , Antagonistas de Receptores de Angiotensina/farmacocinética , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacocinética , Disponibilidade Biológica , Compostos de Bifenilo , Cães , Combinação de Medicamentos , Estabilidade de Medicamentos , Cinética , Preparações Farmacêuticas , Difração de Pó , Solubilidade , Tetrazóis/química , Tetrazóis/farmacocinética , Valsartana/química , Valsartana/farmacocinéticaRESUMO
Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)-Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha-(E)-Dhb. It displays narrow-spectrum activity against vancomycin-resistant Enterococcusâ faecium. In-vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria.
Assuntos
Antibacterianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/química , Alanina/análogos & derivados , Alanina/química , Aminobutiratos/química , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Família Multigênica , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
A single-transaminase-catalyzed biocatalytic cascade was developed by employing the desired biocatalyst, ATA-117-Rd11, that showed high activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinoyl] butyric acid (PPO) and α-ketoglutarate, and low activity against pyruvate. The cascade successfully promotes a highly asymmetric amination reaction for the synthesis of l-phosphinothricin (l-PPT) with high conversion (>95 %) and>99 % ee. In a scale-up experiment, using 10â kg pre-frozen E. coli cells harboring ATA-117-Rd11 as catalyst, 80â kg PPO was converted to ≈70â kg l-PPT after 24â hours with a high ee value (>99 %).
Assuntos
Aminobutiratos/metabolismo , Transaminases/metabolismo , Aminobutiratos/química , Biocatálise , Estrutura MolecularRESUMO
An LC-tandem mass spectrometry method was developed and validated for the simultaneous quantitation of fimasartan and sacubitrilat using positive ion mode. The protein precipitation method was employed for the extraction of fimasartan, sacubitrilat and alprazolam (internal standard) from rat heparinized plasma. Baseline separation of the analytes was accomplished using an ACE-5, C18 (4.6 × 50 mm) column and gradient elution of mobile phase A (5 mm ammonium formate and 0.1% formic acid in purified water) and B (acetonitrile:methanol, 80:20; v/v). All peaks of interest were eluted within a 5-min runtime. The quantitation was achieved in the selected reaction monitoring mode. The developed method was validated as per US Food and Drug Administration guidelines and met the pre-defined acceptance criteria. The method showed linearity from 5 to 10,000 ng/mL. The accuracy/precision of intra- and inter-batch assays was 96.64%/2.05% to 109.17%/13.70% and 100.74%/3.76% to 106.39%/9.75% for fimasartan and 100.02%/1.49% to 113.80%/9.38% and 100.75%/2.31% to 108.40%/7.74% for sacubitrilat, respectively, in rat plasma. Fimasartan and sacubitrilat remained stable in rat plasma at different experimental conditions up to 21 days. The developed method was sensitive, selective and applied successfully to monitor plasma concentrations of fimasartan and sacubitrilat in an oral rat pharmacokinetic study.
Assuntos
Aminobutiratos/sangue , Compostos de Bifenilo/sangue , Cromatografia Líquida/métodos , Pirimidinas/sangue , Espectrometria de Massas em Tandem/métodos , Tetrazóis/sangue , Aminobutiratos/química , Aminobutiratos/farmacocinética , Animais , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Modelos Lineares , Masculino , Pró-Fármacos , Pirimidinas/química , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Tetrazóis/química , Tetrazóis/farmacocinéticaRESUMO
The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.
Assuntos
Proteínas de Bactérias/química , Dioxigenases/química , Glicina/química , Ferro/química , Ácidos Cetoglutáricos/química , Nitrilas/metabolismo , Streptomyces/enzimologia , Aminobutiratos/química , Aminobutiratos/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicina/metabolismo , Histidina/química , Histidina/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Nitrilas/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Streptomyces/química , Streptomyces/genética , Especificidade por Substrato , Vanadatos/química , Vanadatos/metabolismoRESUMO
Ionizable lipids are important compounds of modern therapeutic lipid nano-particles (LNPs). One of the most promising ionizable lipids (or amine lipids) is DLin-MC3-DMA. Depending on their pharmaceutical application these LNPs can also contain various helper lipids, such as phospho- and pegylated lipids, cholesterol and nucleic acids as a cargo. Due to their complex compositions the structures of these therapeutics have not been refined properly. Therefore, the role of each lipid in the pharmacological properties of LNPs has not been determined. In this work an atomistic model for the neutral form of DLin-MC3-DMA was derived and all-atom molecular dynamics (MD) simulations were carried out in order to investigate the effect of the phospholipid headgroup on the possible properties of the shell-membranes of LNPs. Bilayers containing either DOPC or DOPE lipids at two different ratios of DLin-MC3-DMA (5 mol% and 15 mol%) were constructed and simulated at neutral pH 7.4. The results from the analysis of MD trajectories revealed that DOPE lipid headgroups associated strongly with lipid tails and carbonyl oxygens of DLin-MC3-DMA, while for DOPC lipid headgroups no significant associations were observed. Furthermore, the strong associations between DOPE and DLin-MC3-DMA result in the positioning of DLin-MC3-DMA at the surface of the membrane. Such an interplay between the lipids slows down the lateral diffusion of all simulated bilayers, where a more dramatic decrease of the diffusion rate is observed in membranes with DOPE. This can explain the low water penetration of lipid bilayers with phosphatidylethanolamines and, probably, can relate to the bad transfection properties of LNPs with DOPE and DLin-MC3-DMA.
Assuntos
Aminobutiratos/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Microdomínios da Membrana , Simulação de Dinâmica MolecularRESUMO
A chemoenzymatic strategy was developed for the highly efficient synthesis of l-phosphinothricin employing a robust immobilized amidase. An enzymatic hydrolysis of 500 mM N-phenylacetyl-d,l-phosphinothricin resulted in 49.9% conversion and 99.9% ee of l-phosphinothricin within 6 h. To further evaluate the bioprocess for l-phosphinothricin production, the biotransformation was performed for 100 batches under a stirred tank reactor with an average productivity of 8.21 g L-1 h-1. Moreover, unreacted N-phenylacetyl-d-phosphinothricin was racemized and subjected to the enzymatic hydrolysis, giving l-phosphinothricin with a 22.3% yield. A total yield of 69.4% was achieved after one recycle of N-phenylacetyl-d-phosphinothricin. Significantly, this chemoenzymatic approach shows great potential in the industrial production of l-phosphinothricin.
Assuntos
Amidoidrolases/química , Aminobutiratos/química , Proteínas de Bactérias/química , Biotransformação , Enzimas Imobilizadas/química , Hidrólise , CinéticaRESUMO
Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15 400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.
Assuntos
Aminobutiratos/metabolismo , Carboxiliases/metabolismo , Engenharia de Proteínas , Pseudomonas/enzimologia , Aminobutiratos/química , Estrutura MolecularRESUMO
The intrinsic fragility of hydroxyapatite (HAP) restricts its wider applications for local delivery of antibiotics. The composites formed by integrating HAP with hydrogels can improve the properties of HAP. However, these reported composites not only require tedious preparation and employ organic solvent and toxic reagents, but also hardly have inherent antimicrobial property. In this study, N-(9-Fluorenylmethoxycarbonyl)-L-Phenylalanine/nano-hydroxyapatite (Fmoc-L-Phe/nHAP) hybrid supramolecular hydrogels with antibacterial property and cytocompatibility was prepared by integrating nHAP as reinforcement with Fmoc-L-Phe supramolecular hydrogels. The results showed that nHAP bounds in the chamber of the gel network and adheres to the fiber of Fmoc-L-Phe due to intermolecular interaction, remarkably improving the mechanical strength of Fmoc-L-Phe supramolecular hydrogels. The results of inhibition zone experiment and MTT experiment showed that the Fmoc-L-Phe/nHAP hybrid supramolecular hydrogels possess antimicrobial property and cytocompatibility. In vitro release experiment of chlorogenic acid (CGA) from the hybrid supramolecular hydrogels was performed. The study of the release kinetics indicated that the release behavior of CGA from the hybrid supramolecular hydrogels is following Weibull model and release mechanism involved Fickian diffusion and erosion of the surface of hydrogel matrix. The release of CGA shows a good inhibition effect on S. aureus. The results show that the Fmoc-L-Phe/nHAP hybrid hydrogels with antibacterial property and cytocompatibility have promising applications as drug delivery carrier. Due to the intrinsic fragility of hydroxyapatite (HAP), the properties of HAP could be improved by incorporation into hydrogels. However, these reported composites not only require tedious preparation and employ organic solvent and toxic reagents, but also hardly have inherent antimicrobial property. We prepared N-(9-Fluorenylmethoxycarbonyl)-L-Phenylalanine/nano-hydroxyapatite (Fmoc-L-Phe/nHAP) hybrid supramolecular hydrogels by integrating nHAP as reinforcement with Fmoc-L-Phe supramolecular hydrogels. The results showed that nHAP bounds in the chamber of the gel network and adheres to the fiber of Fmoc-L-Phe due to intermolecular interaction, remarkably improving the mechanical strength of Fmoc-L-Phe supramolecular hydrogels. The results of inhibition zone experiment and MTT experiment showed that the Fmoc-L-Phe/nHAP hybrid supramolecular hydrogels possess antibacterial property and cytocompatibility. In vitro release experiment of chlorogenic acid (CGA) from the hybrid supramolecular hydrogels was performed. The study of the release kinetics indicated that the release behavior of CGA from the hybrid supramolecular hydrogels is following Weibull model and release mechanism involved Fickian diffusion and erosion of the surface of hydrogel matrix. The release of CGA shows a good inhibition effect on S. aureus. The results show that the Fmoc-L-Phe/nHAP hybrid hydrogels with antibacterial property and cytocompatibility have promising applications as drug delivery carrier.
Assuntos
Aminobutiratos/química , Portadores de Fármacos , Durapatita/química , Hidrogéis , Aminoácidos/química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Células Cultivadas , Ácido Clorogênico/administração & dosagem , Ácido Clorogênico/farmacocinética , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Excipientes/síntese química , Excipientes/química , Excipientes/farmacologia , Fluorenos/química , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Hidrogéis/farmacologia , Teste de Materiais , Camundongos , Testes de Sensibilidade Microbiana , Nanoestruturas/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Staphylococcus aureusRESUMO
Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.
Assuntos
Aminobutiratos/metabolismo , Proteínas de Bactérias/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico/fisiologia , Actinobacteria/genética , Actinobacteria/metabolismo , Aminobutiratos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Genômica , Família Multigênica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.
Assuntos
Evolução Molecular , Peptídeos/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA/metabolismo , Sequência de Aminoácidos , Aminobutiratos/química , Aminobutiratos/metabolismo , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Origem da Vida , Ornitina/química , Ornitina/metabolismo , Peptídeos/química , Estabilidade Proteica , RNA/química , beta-Alanina/análogos & derivados , beta-Alanina/química , beta-Alanina/metabolismoRESUMO
Herein we present the synthesis of a l-diaminobutanoic acid (DABA)-based nucleopeptide (3), with an oligocationic backbone, realized by solid phase peptide synthesis using thymine-bearing DABA moieties alternating in the sequence with free ones. CD studies evidenced the ability of this oligothymine nucleopeptide, well soluble in aqueous solution, to alter the secondary structure particularly of complementary RNA (poly rA vs poly rU) and inosine-rich RNAs, like poly rI and poly rIC, and showed its preference in binding double vs single-stranded DNAs. Furthermore, ESI mass spectrometry revealed that 3 bound also G-quadruplex (G4) DNAs, with either parallel or antiparallel topologies (adopted in our experimental conditions by c-myc and tel22, respectively). However, it caused detectable changes only in the CD of c-myc (whose parallel G4 structure was also thermally stabilized by ~3 °C), while leaving unaltered the antiparallel structure of tel22. Interestingly, CD and UV analyses suggested that 3 induced a hybrid mixed parallel/antiparallel G4 DNA structure in a random-coil tel22 DNA obtained under salt-free buffer conditions. Titration of the random-coil telomeric DNA with 3 gave quantitative information on the stoichiometry of the obtained complex. Overall, the findings of this work suggest that DABA-based nucleopeptides are synthetic nucleic acid analogues potentially useful in antigene and antisense strategies. Nevertheless, the hexathymine DABA-nucleopeptide shows an interesting behaviour as molecular tool per se thanks to its efficacy in provoking G4 induction in random coil G-rich DNA, as well as for the possibility to bind and stabilize c-myc oncogene in a G4 structure.