Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.792
Filtrar
1.
J Biosci Bioeng ; 130(5): 464-470, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32713813

RESUMO

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.


Assuntos
Monoterpenos Acíclicos/química , Monoterpenos Acíclicos/metabolismo , Ácido Glutâmico/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Synechocystis/metabolismo , Escherichia coli/genética , Aromatizantes/química , Aromatizantes/metabolismo , Engenharia Genética , Ácido Glutâmico/química , Estereoisomerismo , Synechocystis/genética
2.
J Oleo Sci ; 69(8): 865-870, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32641610

RESUMO

We studied the physicochemical properties of 1:1 stoichiometric complexes of acylglutamic acids (CnGlu) with tertiary alkylamines (CnDMA) in water at their low and high concentrations. Static surface tensiometry suggested that the critical micelle concentration (cmc) decreased with increasing hydrophobic chain length of the complexes. In addition, CnGlu-CnDMA yielded lower cmc than the C12Glu single system. In the region of high concentrations, several phase states including isotropic liquid (L1) phase, hexagonal liquid crystal (H1) phase, bicontinuous cubic liquid crystal (V1) phase, and lamellar liquid crystal (Lα) phase were observed. Assemblies with lesser positive curvature tend to be formed with increasing complex concentration, increasing temperature, and increasing hydrophobic chain length. Additionally, the complex formation resulted in the molecular assemblies with lesser positive curvature.


Assuntos
Aminas/química , Fenômenos Químicos , Ácido Glutâmico/química , Tensoativos/química , Água/química , Interações Hidrofóbicas e Hidrofílicas , Cristais Líquidos , Micelas , Tensão Superficial , Temperatura
3.
Int J Nanomedicine ; 15: 2921-2933, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425524

RESUMO

Background: Targeted prodrug has various applications as drug formulation for tumor therapy. Therefore, amphoteric small-molecule prodrug combined with nanoscale characteristics for the self-assembly of the nano-drug delivery system (DDS) is a highly interesting research topic. Methods and Results: In this study, we developed a prodrug self-assembled nanoplatform, 2-glucosamine-fluorescein-5(6)-isothiocyanate-glutamic acid-paclitaxel (2DA-FITC-PTX NPs) by integration of targeted small molecule and nano-DDS with regular structure and perfect targeting ability. 2-glucosamine (DA) and paclitaxel were conjugated as the targeted ligand and anti-tumor chemotherapy drug by amino acid group. 2-DA molecular structure can enhance the targeting ability of prodrug-based 2DA-FITC-PTX NPs and prolong retention time, thereby reducing the toxicity of normal cell/tissue. The fluorescent dye FITC or near-infrared fluorescent dye ICG in prodrug-based DDS was attractive for in vivo optical imaging to study the behavior of 2DA-FITC-PTX NPs. In vitro and in vivo results proved that 2DA-FITC-PTX NPs exhibited excellent targeting ability, anticancer activity, and weak side effects. Conclusion: This work demonstrates a new combination of nanomaterials for chemotherapy and may promote prodrug-based DDS clinical applications in the future.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Paclitaxel/administração & dosagem , Pró-Fármacos/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Fluoresceína-5-Isotiocianato/química , Glucosamina/química , Ácido Glutâmico/química , Humanos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Paclitaxel/química , Paclitaxel/farmacocinética , Pró-Fármacos/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140442, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32376478

RESUMO

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic d-amino acids (i.e., free d-aspartate and d-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than d-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade d-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward d-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded d-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic d-amino acids in biological samples.


Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , D-Aspartato Oxidase/química , Ácido D-Aspártico/química , Flavina-Adenina Dinucleotídeo/química , Ácido Glutâmico/química , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Clonagem Molecular , D-Aspartato Oxidase/genética , D-Aspartato Oxidase/metabolismo , Ácido D-Aspártico/metabolismo , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ácido Glutâmico/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por Substrato
5.
J Chromatogr A ; 1623: 461169, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32376016

RESUMO

Artifacts due to metabolite extraction, derivatization, and detection techniques can result in aberrant observations that are not accurate representations of actual cell metabolism. Here, we show that α-ketoglutarate (α-KG) is reductively aminated to glutamate in methanol:water metabolite extracts, which introduces an artifact into metabolomics studies. We also identify pyridoxamine and urea as amine donors for α-KG to produce glutamate in methanol:water buffer in vitro, and we demonstrate that the addition of ninhydrin to the methanol:water buffer suppresses the reductive amination of α-KG to glutamate in vitro and in metabolite extracts. Finally, we calculate that glutamate levels have been overestimated by 10-50%, depending on cell line, due to α-KG reductive amination. These findings suggest that precautions to account for α-KG reductive amination should be taken for the accurate quantification of glutamate in metabolomics studies.


Assuntos
Artefatos , Ácido Glutâmico/análise , Ácidos Cetoglutáricos/química , Metabolômica , Aminação , Animais , Linhagem Celular , Ácido Glutâmico/química , Metanol/química , Ninidrina/química , Piridoxamina , Ureia/química
6.
J Chromatogr A ; 1621: 461085, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32376018

RESUMO

Two analytical methodologies based on the combined use of hydroxypropyl-ß-cyclodextrin and two different amino acid-based chiral ionic liquids (tetrabutylammonium-L-lysine or tetrabutylammonium-L-glutamic acid) in electrokinetic chromatography were developed in this work to perform the enantioselective determination of econazole and sulconazole in pharmaceutical formulations. The influence of different experimental variables such as buffer concentration, applied voltage, nature and concentration of the ionic liquid, temperature and injection time, on the enantiomeric separation was investigated. The combination of hydroxypropyl-ß-cyclodextrin and tetrabutylammonium-L-lysine under the optimized conditions enabled to achieve the enantiomeric determination of both drugs with high enantiomeric resolution (3.5 for econazole and 2.4 for sulconazole). The analytical characteristics of the developed methodologies were evaluated in terms of linearity, precision, LOD, LOQ and recovery showing good performance for the determination of both drugs which were successfully quantitated in pharmaceutical formulations. This work reports the first analytical methodology enabling the enantiomeric determination of sulconazole in pharmaceutical formulations.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Cromatografia Capilar Eletrocinética Micelar/métodos , Econazol/análise , Ácido Glutâmico/química , Imidazóis/análise , Líquidos Iônicos/química , Lisina/química , Compostos de Amônio Quaternário , Estereoisomerismo , Temperatura , Fatores de Tempo
7.
Arch Biochem Biophys ; 685: 108348, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32198047

RESUMO

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ácido Glutâmico/química , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Calmodulina/metabolismo , Domínio Catalítico , Células HEK293 , Humanos , Cinética , Mutação , Ligação Proteica
8.
J Mol Model ; 26(4): 74, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32146585

RESUMO

Protein-based electronics is one of the emerging technology in which inventive electronic devices are being adduced and developed based on the selective actions of specific proteins. The explicit actions can be predicted if the building blocks of proteins (i.e., amino acids) are studied decorously. We emphasize our work on electronic transport properties of L-glutamic acid (i.e., L-amino acid) stringed to gold, silver, and copper electrodes, respectively, to form three distinct devices. For our calculations, we employ NEGF-DFT approach using self-consistent function. Electronic coupling and tunneling barriers between the molecule and the electrodes have been emphasized with an inception of delocalization of molecular orbitals within the device. We observe strong correlation between tunneling barrier and Mulliken charge transfer between molecule and electrodes. The asymmetrical carbon chain (-CH2) within the molecule exhibits negative differential resistance (NDR) and rectification ratio. The device using molecule with copper electrodes exhibits the highest peak to valley current ratio of 1.84. The rectification ratio of the device with gold, silver, and copper electrodes is 2.35, 2.25, and 15.62, respectively, at finite bias. These results yield fresh insight on the potential of L-glutamic acid like bio-molecule in the emerging field of proteotronics.


Assuntos
Eletrônica , Ácido Glutâmico/química , Metais/química , Eletrodos
9.
Proc Natl Acad Sci U S A ; 117(7): 3839-3847, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015122

RESUMO

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.


Assuntos
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Dimerização , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ratos , Receptores de N-Metil-D-Aspartato/genética
11.
Molecules ; 25(4)2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32079282

RESUMO

Cubozoan nematocyst venoms contain known cytolytic and hemolytic proteins, but small molecule components have not been previously reported from cubozoan venom. We screened nematocyst extracts of Alatina alata and Chironex yamaguchii by LC-MS for the presence of small molecule metabolites. Three isomeric compounds, cnidarins 4A (1), 4B (2), and 4C (3), were isolated from venom extracts and characterized by NMR and MS, which revealed their planar structure as cyclic γ-linked tetraglutamic acids. The full configurational assignments were established by syntheses of all six possible stereoisomers, comparison of spectral data and optical rotations, and stereochemical analysis of derivatized degradation products. Compounds 1-3 were subsequently detected by LC-MS in tissues of eight other cnidarian species. The most abundant of these compounds, cnidarin 4A (1), showed no mammalian cell toxicity or hemolytic activity, which may suggest a role for these cyclic tetraglutamates in nematocyst discharge.


Assuntos
Cubomedusas/química , Ácido Glutâmico/biossíntese , Ácido Glutâmico/isolamento & purificação , Animais , Organismos Aquáticos/química , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Venenos de Cnidários/química , Venenos de Cnidários/toxicidade , Ácido Glutâmico/química , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Distribuição Tecidual
12.
J Mol Biol ; 432(7): 2186-2203, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32087198

RESUMO

3,6-anhydro-α-L-galactose (L-AHG) is one of the main monosaccharide constituents of red macroalgae. In the recently discovered bacterial L-AHG catabolic pathway, L-AHG is first oxidized by a NAD(P)+-dependent dehydrogenase (AHGD), which is a key step of this pathway. However, the catalytic mechanism(s) of AHGDs is still unclear. Here, we identified and characterized an AHGD from marine bacterium Vibrio variabilis JCM 19239 (VvAHGD). The NADP+-dependent VvAHGD could efficiently oxidize L-AHG. Phylogenetic analysis suggested that VvAHGD and its homologs represent a new aldehyde dehydrogenase (ALDH) family with different substrate preferences from reported ALDH families, named the L-AHGDH family. To explain the catalytic mechanism of VvAHGD, we solved the structures of VvAHGD in the apo form and complex with NADP+ and modeled its structure with L-AHG. Based on structural, mutational, and biochemical analyses, the cofactor channel and the substrate channel of VvAHGD are identified, and the key residues involved in the binding of NADP+ and L-AHG and the catalysis are revealed. VvAHGD performs catalysis by controlling the consecutive connection and interruption of the cofactor channel and the substrate channel via the conformational changes of its two catalytic residues Cys282 and Glu248. Comparative analyses of structures and enzyme kinetics revealed that differences in the substrate channels (in shape, size, electrostatic surface, and residue composition) lead to the different substrate preferences of VvAHGD from other ALDHs. This study on VvAHGD sheds light on the diversified catalytic mechanisms and evolution of NAD(P)+-dependent ALDHs.


Assuntos
Cisteína/química , Galactose Desidrogenases/metabolismo , Galactose/análogos & derivados , Ácido Glutâmico/química , NADP/metabolismo , Vibrio/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cisteína/genética , Cisteína/metabolismo , Galactose/metabolismo , Galactose Desidrogenases/química , Galactose Desidrogenases/genética , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Modelos Moleculares , Mutação , Filogenia , Homologia de Sequência
13.
Phys Chem Chem Phys ; 22(5): 3008-3016, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957772

RESUMO

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Proteínas/química , Espectrofotometria Infravermelho , Ácido Aspártico/química , Ácido Glutâmico/química , Teoria Quântica
14.
Nat Commun ; 11(1): 423, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969570

RESUMO

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition.


Assuntos
Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Animais , Sítios de Ligação , Ligação Competitiva , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Cinética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Xenopus laevis
15.
Anal Chim Acta ; 1099: 119-125, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-31986268

RESUMO

γ-Glutamyl transpeptidase (GGT) has attracted considerable attention for its regulatory effect on glutathione metabolism in living organisms; further, its close relationship with physiological dysfunctions such as hepatitis and liver cancers has enhanced its applicability. Therefore, the accurate detection of GGT levels is particularly important for the early diagnosis of diseases. Thus, we herein report the development of a surface-enhanced Raman spectroscopic (SERS) probe, namely bis-s,s'-((s)-4,4'-thiolphenylamide-Glu) (b-(s)-TPA-Glu), that comprises of a γ-glutamyl moiety for detection of the GGT activity. In this system, detection was achieved by observing differences in the SERS spectral profiles of the b-(s)-TPA-Glu probe and its corresponding hydrolysis product that resulted from the catalytic action of GGT. This SERS probe system exhibited a high selectivity toward GGT due to a combination of its specific catalytic action and the distinctive spectroscopic fingerprint of the SERS technique. The developed SERS approach was also found to be approximately linear in the range of 0.2-200 U/L, and a limit of detection of 0.09 U/L was determined. Furthermore, the proposed SERS method was suitable for detection of the GGT activity of clinical serum samples and also for evaluation of the inhibitors of GGT. Consequently, this approach is considered to be a promising diagnostic and drug screening tool for GGT-associated diseases.


Assuntos
Sondas Moleculares/química , gama-Glutamiltransferase/sangue , Amidas/química , Ácido Glutâmico/química , Humanos , Estrutura Molecular , Nanopartículas/química , Prata/química , Análise Espectral Raman , Compostos de Sulfidrila/química , Propriedades de Superfície , gama-Glutamiltransferase/metabolismo
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118059, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-32000059

RESUMO

DL-glutamic acid monohydrate crystal was synthesized from an aqueous solution by slow evaporation technique. The crystal was submitted to high-pressure (1 atm-14.3 GPa) to investigate its vibrational behavior and the occurrence of phase transitions. We performed Raman spectroscopy as probe and through the analysis of the spectra we discovered three structural phase transitions. The first one occurs around 0.9 GPa. In this phase transition, glutamic acid molecules suffer modifications in their conformations while water molecules are less affected. The second phase transition at 4.8 GPa involves conformational changes related to CO2-, NH3+ units and the water molecules, while the third one, between 10.9 and 12.4 GPa, involves motions of several parts of the glutamic acid as well as the water molecules. Considering the dynamic of high pressure, the second phase of DL-glutamic acid monohydrate crystal presented a better stability compared with the second phase of its polymorphs α and ß L-glutamic acid. In addition, water molecules seem to play important role on this structural stability. All changes are reversible.


Assuntos
Ácido Glutâmico/química , Conformação Molecular , Transição de Fase , Pressão , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Análise Espectral Raman
17.
Biosci Biotechnol Biochem ; 84(1): 118-125, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516066

RESUMO

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.


Assuntos
4-Aminobutirato Transaminase/química , Aminoácido Oxirredutases/química , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Peroxidase/química , Ácido gama-Aminobutírico/análise , Ampirona/química , Ascorbato Oxidase/química , Catalase/química , Cromatografia Líquida , Ensaios Enzimáticos/economia , Compostos Férricos/química , Ácido Glutâmico/química , Peróxido de Hidrogênio/química , Lycopersicon esculentum/química , Acoplamento Oxidativo , Proteínas Recombinantes , Streptomyces/enzimologia , Espectrometria de Massas em Tandem
18.
J Biol Chem ; 295(2): 435-443, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767681

RESUMO

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.


Assuntos
Espastina/metabolismo , Tubulina (Proteína)/metabolismo , Sítios de Ligação , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espastina/química , Tubulina (Proteína)/química
19.
Eur J Mass Spectrom (Chichester) ; 26(3): 175-186, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31874577

RESUMO

Fragmentation mechanisms of the singly protonated glutathione (γ-ECG) and its synthetic analogue peptides (ECG and PPECG) have been investigated by liquid chromatography tandem-mass spectrometry and theoretical calculations. In the mass spectra, similar fragmentation patterns were observed for γ-ECG and ECG, but a completely different one was found in the case of PPECG. The E-C amide bond cleavage is the predominant pathway for the fragmentation of γ-ECG and ECG, whereas the additional N-terminal prolyl residues in PPECG significantly suppress the E-C amide bond cleavage. Theoretical calculations reveal that the fragmentation efficiencies of the E-C bonds in the protonated γ-ECG and ECG are much higher than that in the protonated PPECG, being attributed to their lower barriers of the potential energy; clearly the introduction of two prolyl residues can increase substantially the potential energy barrier. In the proposed mechanism, the protonated E-C amide bonds in the three peptides are first weakened followed by a nucleophilic addition by the glutamyl carboxyl oxygen atom in side chain, leading to the breaking of the E-C amide bonds. However, the processes of E-C bond fragmentation for three protonated analogs were not collaborative. Protonated amide bonds first fragment, then the nucleophilic addition by the side chain of glutamyl carboxyl oxygen atom takes places. On the other hand, the prolyl residues in PPECG can largely diminish the nucleophilic addition, resulting in a much lower efficiency of its E-C amide bond breaking. Distance analysis indicates that breaking the E-C amide bonds in the protonated γ-ECG, ECG, and PPECG ions could not occur without the assistance from the nucleophilic attack, highlighting an asynchronous collaborative process in the bond breakings.


Assuntos
Ácido Glutâmico/química , Glutationa/química , Peptídeos/química , Íons/química , Espectrometria de Massas , Estrutura Molecular
20.
Amino Acids ; 51(10-12): 1609-1621, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31712920

RESUMO

The role of pH-dependent ionic structures of L-amino acids in catalysis has been investigated for the two-component Mannich reactions between dimethyl malonate (DMM)/ethyl acetoacetate (EAA) and imines. As catalysts, L-amino acids performed well, even better than corresponding base catalysts and provided the ß-amino carbonyl compounds in very high yields. Density functional calculations were used to gain the mechanistic insight of the reaction. High catalytic efficiency of amino acids was attributed to the facile formation of carbanion intermediate through barrierless transition state TS1 (- 19.43 kcal/mol) and then its stabilization owing to carbanion interaction with protonated amino acid.


Assuntos
Aminoácidos/química , Acetoacetatos/química , Catálise , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Iminas/química , Malonatos/química , Bases de Mannich/síntese química , Bases de Mannich/química , Estrutura Molecular , Estereoisomerismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA