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1.
Hum Genet ; 138(10): 1183-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31471722

RESUMO

The glutamate pyruvate transaminase 2 (GPT2) gene produces a nuclear-encoded mitochondrial enzyme that catalyzes the reversible transfer of an amino group from glutamate to pyruvate, generating alanine and alpha-ketoglutarate. Recessive mutations in GPT2 have been recently identified in a new syndrome involving intellectual and developmental disability (IDD), postnatal microcephaly, and spastic paraplegia. We have identified additional families with recessive GPT2 mutations and expanded the phenotype to include small stature. GPT2 loss-of-function mutations were identified in four families, nine patients total, including: a homozygous mutation in one child [c.775T>C (p.C259R)]; compound heterozygous mutations in two siblings [c.812A>C (p.N271T)/c.1432_1433delGT (p.V478Rfs*73)]; a novel homozygous, putative splicing mutation [c.1035C>T (p.G345=)]; and finally, a recurrent mutation, previously identified in a distinct family [c.1210C>T (p.R404*)]. All patients were diagnosed with IDD. A majority of patients had remarkably small stature throughout development, many < 1st percentile for height and weight. Given the potential biological function of GPT2 in cellular growth, this phenotype is strongly suggestive of a newly identified clinical susceptibility. Further, homozygous GPT2 mutations manifested in at least 2 of 176 families with IDD (approximately 1.1%) in a Pakistani cohort, thereby representing a relatively common cause of recessive IDD in this population, with recurrence of the p.R404* mutation in this population. Based on variants in the ExAC database, we estimated that approximately 1 in 248 individuals are carriers of moderately or severely deleterious variants in GPT2.


Assuntos
Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Genes Recessivos , Predisposição Genética para Doença , Mutação , Fenótipo , Transaminases/genética , Adolescente , Alelos , Substituição de Aminoácidos , Deficiências do Desenvolvimento/metabolismo , Ativação Enzimática , Éxons , Feminino , Frequência do Gene , Estudos de Associação Genética , Genética Populacional , Genótipo , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Imagem por Ressonância Magnética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Linhagem , Conformação Proteica , Sítios de Splice de RNA , Análise de Sequência de DNA , Relação Estrutura-Atividade , Transaminases/química , Transaminases/metabolismo
2.
J Med Life ; 12(2): 168-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406519

RESUMO

There is evidence that infection by H. pylori can have a critical proportion in the development of hepatocyte injury and both noncancerous and malignant liver conditions including non-alcoholic fatty liver disease (NAFLD). This is attributed to several mechanisms, the most important one being the toxic products of the bacterium H. pylori and oxidative injury for hepatocytes which promotes hepatic injury. The present research was aimed at determining the association between H. pylori infection and the prevalence of NAFLD in Birjand, Iran. Two groups were included in this cross-sectional study at the outpatient university clinic. One group had NAFLD (65 patients) and the other group was healthy controls without NAFLD (65 subjects). The diagnosis of NAFLD was performed using abdominal ultrasound examination and the absence of taking steatogenic medications or alcohol. Serum anti-H. pylori IgG and fecal H. pylori antigen were tested for diagnosing of H. pylori infection using ELISA method. H. pylori infection diagnosis was made if both tests were positive. None of the subjects in either group had symptoms related to the digestive system including dyspepsia, GERD (gastroesophageal reflux disease), or epigastric pain suspicious of peptic ulcer disease. There were 37 patients (28.5%) in both NAFLD (22 cases, 33.8%) and control (15 cases, 23.1%) groups whose H. pylori tests (both IgG and fecal antigen) were positive. Statistically, no significant difference was observed between the two studied groups regarding H. pylori infection frequency (p = 0.37). Asymptomatic H. pylori infection rate was not significantly different between NAFLD patients and control subjects in Birjand, Iran.


Assuntos
Infecções por Helicobacter/complicações , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/fisiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Adulto , Estudos de Casos e Controles , Estudos Transversais , Feminino , Infecções por Helicobacter/sangue , Humanos , Irã (Geográfico)/epidemiologia , Lipídeos/sangue , Fígado/enzimologia , Fígado/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica/sangue , Transaminases/metabolismo
3.
Pestic Biochem Physiol ; 158: 156-165, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378352

RESUMO

Culex pipiens is a main vector for Bancroftian filariasis, Rift Valley Fever and diseases caused by other viruses, leaving several peoples with disabilities. In recent years, plant derived compounds have received much attention as potential alternatives to synthetic chemicals due to their low toxicity to mammals and environmental persistence. Twenty-one monoterpenes from different chemical groups (hydrocarbons and oxygenated products) were evaluated against Culex pipiens larvae. In addition, in vivo biochemical studies including effects on acetylcholine esterase (AChE), acid and alkaline phosphatases (ACP and ALP), total adenosine triphosphatase (ATPase) and gamma-aminobutyric acid transaminase (GABA-T) were investigated. Furthermore, in silico studies including pharmacophore elucidation, ADMET analysis and molecular docking of these compounds were performed. Among all tested monoterpenes, hydrocarbons [p-cymene, (R)-(+)-limonene and (+)-α-pinene], acetates (cinnamyl acetate, citronellyl acetate, eugenyl acetate and terpinyl acetate), alcohols [(±)-ß-citronellol and terpineol], aldehydes [citral and (1R)-(-)-myrtenal] and ketone [(R)-(+)-pulegone] exhibited the highest larval toxicity with LC50 = 14.88, 27.97, 26.13, 2.62, 3.81, 2.74, 21.65, 1.64, 21.70, 21.76, 1.68 and 1.90 mg/L after 48 h of exposure, respectively. The compounds proved a significant inhibition of all tested enzymes except total ATPase. The biochemical and molecular docking studies proved that AChE and GABA-T were the main targets for the tested monoterpenes.


Assuntos
Culex/virologia , Inseticidas/farmacologia , Monoterpenos/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Culex/patogenicidade , Filariose Linfática/transmissão , Ativação Enzimática/efeitos dos fármacos , Esterases/metabolismo , Simulação de Acoplamento Molecular , Transaminases/metabolismo
4.
Food Chem ; 297: 125035, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253330

RESUMO

In this study, an electrochemical system was established to detect the branched-chain amino acid aminotransferase (BCAT) activity in lactic acid bacteria (LAB). A nanocomposite of chitosan (CS) with multi-walled carbon nanotubes (MWCNTs) was synthesized, and the composite solution were uniformly spread over the glassy carbon electrode (GCE) surface by drop-casting to fabricate an electrochemical biosensor. The composite was characterized by scanning electron microscopy (SEM) and cyclic voltammetry (TEM). Results indicated that the MWCNTs-CS/GCE electrode exhibited higher stability and sensitivity, compared with the GCE electrode. The linear response for nicotinamide adenine dinucleotide (NADH) was 1.0-9.0 µM and the response limit was 0.12 µM. The system effectively and sensitively detected the BCAT activity by NADH concentration in the LAB culture, comparing with the optical method. The culture condition of LAB was optimized by using this system, evidencing that established method was available to detect the BCAT activity of LAB.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Eletroquímicas/métodos , Lactobacillales/enzimologia , Transaminases/metabolismo , Técnicas Biossensoriais/métodos , Quitosana/química , Eletrodos , Proteínas Musculares/metabolismo , NAD/química , NAD/metabolismo , Nanotubos de Carbono/química
5.
Nat Commun ; 10(1): 2767, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235694

RESUMO

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications.


Assuntos
Metabolismo Energético/fisiologia , Fadiga/fisiopatologia , Cinurenina/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Adaptação Fisiológica , Animais , Aspartato Aminotransferases/metabolismo , Ácido Aspártico/metabolismo , Carbidopa/farmacologia , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Glicólise/fisiologia , Malatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Animais , Músculo Esquelético/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Condicionamento Físico Animal/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transaminases/antagonistas & inibidores , Transaminases/metabolismo
6.
Plant Cell Rep ; 38(9): 1165-1180, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31161264

RESUMO

KEY MESSAGE: Arabidopsis photorespiratory gene AtAGT1 is important for the growth and development of root, the non-photosynthetic organ, and it is involved in a complex metabolic network and salt resistance. AtAGT1 in Arabidopsis encodes an aminotransferase that has a wide range of donor:acceptor combinations, including Asn:glyoxylate. Although it is one of the photorespiratory genes, its encoding protein has been suggested to function also in roots to metabolize Asn. However, experimental data are still lacking. In this study, we investigated experimentally the function of AtAGT1 in roots and our results uncovered its importance in root development during seedling establishment after seed germination. Overexpression of AtAGT1 in roots promoted both the growth of primary root and outgrowth of lateral roots. To further elucidate the molecular mechanisms underlying, amino acid content and gene expression in roots were analyzed, and results revealed that AtAGT1 is involved in a complex metabolic network and salt resistance of roots.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Expressão Gênica , Germinação , Plantas Geneticamente Modificadas , Tolerância ao Sal , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia , Transaminases/genética , Transaminases/metabolismo
7.
Food Chem ; 294: 267-275, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126462

RESUMO

Escherichia coli was engineered to produce d-pantothenic acid via systematic metabolic engineering. Firstly, genes of acetohydroxy acid synthase II, pantothenate synthetase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, 2-dehydropantoate 2-reductase and ketol-acid reductoisomerase were edited in E. coli W3110 with a resulting d-pantothenic acid yield of 0.49 g/L. Expressions of valine-pyruvate aminotransferase and branched-chain-amino-acid aminotransferase were then attenuated to decrease the carbon flux in l-valine biosynthetic pathway which is a competing pathway to the d-pantothenic acid biosynthetic pathway, and the yield increased to 1.48 g/L. Mutagenesis of pantothenate kinase and deletion of threonine deaminase further increased the production to 1.78 g/L. Overexpressions of panC and panB from Corynebacterium glutamicum enhanced the production by 29%. In fed-batch fermentations, strain DPA-9/pTrc99a-panBC(C.G) exhibited a highest d-pantothenic acid yield of 28.45 g/L. The findings in this study demonstrate the systematic metabolic engineering in Escherichia coli W3110 would be a promising strategy for industrial production of d-pantothenic acid.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Ácido Pantotênico/biossíntese , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Corynebacterium glutamicum/genética , Escherichia coli/crescimento & desenvolvimento , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Mutagênese , Ácido Pantotênico/química , Transaminases/genética , Transaminases/metabolismo , Valina/biossíntese
8.
Int J Biol Macromol ; 132: 1012-1023, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30959130

RESUMO

Phosphoserine aminotransferase (PSAT) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the second reversible step in the phosphoserine biosynthetic pathway producing serine. The crystal structure of E. histolytica PSAT (EhPSAT) complexed with PLP was elucidated at 3.0 Šresolution and the structures of its mutants, EhPSAT_Δ45 and EhPSAT_Δ4, at 1.8 and 2.4 Šresolution respectively. Deletion of 45 N-terminal residues (EhPSAT_Δ45) resulted in an inactive protein, the structure showed a dimeric arrangement drastically different from that of the wild-type protein, with the two monomers translated and rotated by almost 180° with respect to each other; causing a rearrangement of the active site to which PLP was unable to bind. Deletion of first N-terminal 15 (EhPSAT_Δ15) and four 11th to 14th residues (EhPSAT_Δ4) yielded up to 98% and 90% decrease in the activity respectively. Absence of aldimine linkage between PLP-Lys in the crystal structure of EhPSAT_Δ4 mutant explains for such decrease in activity and describes the importance of these N-terminal residues. Furthermore, a halide-binding site was found in close proximity to the active site. A stretch of six amino acids (146-NNTIYG-151) only conserved in the Entamoeba genus, contributes to halide binding may explain that the halide inhibition could be specific to Entamoeba.


Assuntos
Domínio Catalítico , Entamoeba histolytica/enzimologia , Transaminases/química , Transaminases/metabolismo , Sequência de Aminoácidos , Cloretos/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Estrutura Quaternária de Proteína , Análise de Sequência , Transaminases/genética
9.
Molecules ; 24(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934681

RESUMO

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.


Assuntos
Aspergillus/fisiologia , Mutação , Transaminases/metabolismo , Aminas/química , Aminas/metabolismo , Estabilidade Enzimática , Cinética , Conformação Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Termodinâmica , Transaminases/química
10.
Arch Insect Biochem Physiol ; 101(2): e21553, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004387

RESUMO

In this study, we identified and characterized a phosphoserine aminotransferase (bmPSAT) from Bombyx mori (B. mori) that is responsible for l-serine biosynthesis. A complementary DNA that encodes bmPSAT was cloned by reverse transcriptase polymerase reaction and sequenced. The presumed amino acid sequence revealed 47-87% identity with known PSATs from insects, humans, plants, and bacteria. Through phylogenetic analysis, we found that bmPSAT is evolutionary related to insect PSATs. Recombinant bmPSAT was produced in Escherichia coli by using a cold-shock promotor and purified to homogeneity. This enzyme utilizes phosphohydroxypyruvate and glutamate for transamination. bmPSAT messenger RNA (mRNA) was expressed at higher levels in several tissues of standard strain silkworm including the silk gland, whereas a sericin-deficient silkworm strain exhibited a diminished expression of bmPSAT mRNA in the silk gland. These findings indicate that bmPSAT may play an important role in synthesizing and supplying l-serine in the larva of B. mori.


Assuntos
Bombyx/enzimologia , Serina/biossíntese , Transaminases/química , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Transaminases/genética , Transaminases/metabolismo
11.
Biochim Biophys Acta Proteins Proteom ; 1867(6): 575-585, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30902765

RESUMO

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-α-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0-10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 Šresolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards α-ketoglutarate and increase the affinity towards (R)-(+)-α-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.


Assuntos
Myxococcales/enzimologia , Transaminases/química , Transaminases/metabolismo , Aminoácidos de Cadeia Ramificada , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos , Fenetilaminas/metabolismo , Estresse Salino
12.
BMC Med Genet ; 20(1): 56, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925902

RESUMO

BACKGROUND: PHKA2 gene mutations can cause liver phosphorylase kinase (PhK) deficiency, resulting in glycogen storage disease type IXa (GSD IXa). Elevated liver transaminase levels and liver enlargement are the most frequent phenotypes of GSD IXa. However, whether the phenotypes are applicable to Chinese patients remains unclear. CASE REPORT: A boy aged 2 years and 8 months with a history of episodic fatigue and weakness since he was 2 years old was referred to our endocrinology clinic. Apart from symptomatic ketotic hypoglycemic episodes (palpitation, hand shaking, sweating, etc.), no abnormalities of liver transaminase levels or liver size were found. To identify the aetiology of his clinically diagnosed hypoglycaemia, the proband and his parents were screened for PHKA2 gene mutations by next-generation sequencing. A heterozygous mutation (c.2972C > G, p.G991A) in PHKA2 was found in the proband and his mother. Twenty-one Chinese cases with GSD IXa have been reported in the literature to date, and elevated liver transaminase levels (95%) and liver enlargement (91%) are the most frequent phenotypes of GSD IXa in Chinese patients. Hypoglycaemia may be one of the early onset symptoms in infants with GSD IXa. CONCLUSIONS: This study enriches our knowledge of the PHKA2 gene mutation spectrum and provides further information about the phenotypic characteristics of Chinese GSD IXa patients.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Doença de Depósito de Glicogênio/genética , Hipoglicemia/complicações , Fosforilase Quinase/genética , Mutação Puntual , Pré-Escolar , Doença de Depósito de Glicogênio/etiologia , Doença de Depósito de Glicogênio/metabolismo , Doença de Depósito de Glicogênio/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hipoglicemia/genética , Fígado/enzimologia , Fígado/patologia , Masculino , Linhagem , Fenótipo , Análise de Sequência de DNA , Transaminases/metabolismo
13.
Nanoscale ; 11(14): 6990-7001, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916672

RESUMO

Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo. We confirmed uptake of EVs derived from Expi293F cells into HepG2 cells and did not detect any signs of hepatotoxicity measured by cell viability, functional secretion of albumin, plasma membrane integrity, and mitochondrial and lysosomal activity even at high exposures of up to 5 × 1010 EVs per mL. Whole genome transcriptome analysis was used to measure potential effects on the gene expression in the recipient HepG2 cells at 24 h following exposure to EVs. Only 0.6% of all genes were found to be differentially expressed displaying less than 2-fold expression change, with genes related to inflammation or toxicity being unaffected. EVs did not trigger any proinflammatory cytokine response in HepG2 cells. However, minor changes were noted in human blood for interleukin (IL)-8, IL-6, and monocyte chemotactic protein 1 (MCP-1). Administration of 5 × 1010 Expi293F-derived EVs to BALB/c mice did not result in any histopathological changes or increases of liver transaminases or cytokine levels, apart from a modest increase in keratinocyte chemoattractant (KC). The absence of any significant toxicity associated with EVs in vitro and in vivo supports the prospective use of EVs for therapeutic applications and for drug delivery.


Assuntos
Vesículas Extracelulares/fisiologia , Fígado/patologia , Animais , Citocinas/metabolismo , Vesículas Extracelulares/transplante , Células HEK293 , Células Hep G2 , Humanos , Mediadores da Inflamação/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Albumina Sérica/metabolismo , Transaminases/metabolismo , Transcriptoma
14.
J Biotechnol ; 295: 49-54, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30853639

RESUMO

(R)-3-amino-1-butanol is a key intermediate of Dolutegravir for the treatment of HIV/AIDS and its green and efficient biosynthesis has attracted a great deal of attention. Transaminases are currently used as promising biocatalyst for the synthesis of chiral amines. However, many transaminases have (S)-specificity and (R)-selective transaminases were less exploited and studied, making the production of (R)-amines remain challenging. In this study, a novel transaminase from Actinobacteria sp. (As-TA) was obtained and applied for the biosynthesis of (R)-3-amino-1-butanol by transferring the amino group from isopropylamine to 4-hydroxy-2-butanone. After optimization of the reaction condition and using a substrate fed-batch strategy, the conversion of 100, 200, 300, 400 and 500 mM 4-hydroxy-2-butanone reached 100%, 94.9%, 86.1%, 76.1% and 70.9%, respectively. (R)-3-amino-1-butanol with a maximum yield of 29.6 g/L and 99.9% e.e. value was obtained. This was the first time demonstrating the successful biosynthesis of (R)-3-amino-1-butanol with transaminase as biocatalyst and the obtained As-TA enriched the enzyme pool of transaminase with (R)-specificity.


Assuntos
Actinobacteria/enzimologia , Amino Álcoois/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/metabolismo , Transaminases/metabolismo , Actinobacteria/genética , Amino Álcoois/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biotecnologia , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Engenharia Metabólica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Temperatura Ambiente , Transaminases/química , Transaminases/genética
15.
Mar Drugs ; 17(3)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836614

RESUMO

Diazobenzofluorene-containing atypical angucyclines exhibit promising biological activities. Here we report the inactivation of an amidotransferase-encoding gene flsN3 in Micromonospora rosaria SCSIO N160, a producer of fluostatins. Bioinformatics analysis indicated that FlsN3 was involved in the diazo formation. Chemical investigation of the flsN3-inactivation mutant resulted in the isolation of a variety of angucycline aromatic polyketides, including four racemic aminobenzo[b]fluorenes stealthins D⁻G (9⁻12) harboring a stealthin C-like core skeleton with an acetone or butanone-like side chain. Their structures were elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopic data and X-ray diffraction analysis. A plausible mechanism for the formation of stealthins D⁻G (9⁻12) was proposed. These results suggested a functional role of FlsN3 in the formation/modification of N⁻N bond-containing fluostatins.


Assuntos
Organismos Aquáticos/metabolismo , Proteínas de Bactérias/metabolismo , Fluorenos/isolamento & purificação , Micromonospora/metabolismo , Transaminases/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Biologia Computacional , Cristalografia por Raios X , Fluorenos/química , Fluorenos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Nitrogênio/química , Streptomyces , Transaminases/genética
16.
Int J Mol Sci ; 20(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857183

RESUMO

The aminotransferase from Bacillus circulans (BtrR), which is involved in the biosynthesis of butirosin, catalyzes the pyridoxal phosphate (PLP)-dependent transamination reaction to convert valienone to ß-valienamine (a new ß-glycosidase inhibitor for the treatment of lysosomal storage diseases) with an optical purity enantiomeric excess value. To explore the stereoselective mechanism of valienamine generated by BtrR, multiple molecular dynamics (MD) simulations were performed for the BtrR/PLP/valienamine and BtrR/PLP/ß-valienamine complexes. The theoretical results showed that ß-valienamine could make BtrR more stable and dense than valienamine. ß-valienamine could increase the hydrogen bond probability and decrease the binding free energy between coenzyme PLP and BtrR by regulating the protein structure of BtrR, which was conducive to the catalytic reaction. ß-valienamine maintained the formation of cation-p interactions between basic and aromatic amino acids in BtrR, thus enhancing its stability and catalytic activity. In addition, CAVER 3.0 analysis revealed that ß-valienamine could make the tunnel of BtrR wider and straight, which was propitious to the removal of products from BtrR. Steered MD simulation results showed that valienamine interacted with more residues in the tunnel during dissociation compared with ß-valienamine, resulting in the need for a stronger force to be acquired from BtrR. Taken together, BtrR was more inclined to catalyze the substrates to form ß-valienamine, either from the point of view of the catalytic reaction or product removal.


Assuntos
Bacillus/metabolismo , Cicloexenos/metabolismo , Hexosaminas/metabolismo , Simulação de Dinâmica Molecular , Transaminases/metabolismo , Bacillus/química , Bacillus/enzimologia , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Fosfato de Piridoxal/metabolismo , Estereoisomerismo , Especificidade por Substrato , Transaminases/química
17.
Pharmacol Res Perspect ; 7(1): e00467, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30784208

RESUMO

During a randomized Phase 1 clinical trial the drug candidate, PF-04895162 (ICA-105665), caused transaminase elevations (≥grade 1) in six of eight healthy subjects treated at 300 mg twice daily for 2-weeks (NCT01691274). This was unexpected since studies in rats (<6 months) and cynomolgus monkeys (<9 months) treated up to 100 mg/kg/day did not identify the liver as a target organ. Mechanistic studies showed PF-04895162 had low cytotoxic potential in human hepatocytes, but inhibited liver mitochondrial function and bile salt export protein (BSEP) transport. Clinical relevance of these postulated mechanisms of liver injury was explored in three treated subjects that consented to analysis of residual pharmacokinetic plasma samples. Compared to a nonresponder, two subjects with transaminase elevations displayed higher levels of miRNA122 and total/conjugated bile acid species, whereas one demonstrated impaired postprandial clearance of systemic bile acids. Elevated taurine and glycine conjugated to unconjugated bile acid ratios were observed in two subjects, one before the onset of elevated transaminases. Based on the affinity of conjugated bile acid species for transport by BSEP, the profile of plasma conjugated/unconjugated bile acid species was consistent with inhibition of BSEP. These data collectively suggest that the human liver injury by PF-04895162 was due to alterations in bile acid handling driven by dual BSEP/mitochondrial inhibition, two important risk factors associated with drug-induced liver injury in humans. Alterations in systemic bile acid composition were more important than total bile acids in the manifestation of clinical liver injury and may be a very early biomarker of BSEP inhibition.


Assuntos
Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Adulto , Animais , Transporte Biológico/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Método Duplo-Cego , Hepatócitos/metabolismo , Homeostase , Humanos , Macaca fascicularis , Masculino , Mitocôndrias Hepáticas/metabolismo , Ratos , Fatores de Risco , Especificidade da Espécie , Transaminases/metabolismo , Adulto Jovem
18.
J Affect Disord ; 245: 1119-1125, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30699855

RESUMO

OBJECTIVE: Major depressive disorder (MDD) is a complex mental disorder. The lack of well-established biomarkers hinders its diagnosis, treatment, and new-drug development. N-methyl-D-aspartate receptor (NMDAR) dysfunction has been implicated in the pathogenesis of MDD. This study examined whether expressions of the NMDAR-related genes are characteristic of MDD. METHODS: Expressions of NMDAR-related genes including SRR, SHMT2, PSAT1, GCAT, GAD1, SLC1A4, NRG1 and COMT in peripheral WBCs of 110 patients with MDD (25 drug-naïve, 21 drug-free, and 64 medicated patients) and 125 healthy individuals were measured using quantitative PCR. RESULTS: The mRNA expression levels of SRR, PSAT1, GCAT, GAD1, NRG1 and COMT were significantly different among the four groups (all p < 0.05). For drug-naïve patients, the ΔΔCT values of SRR, PSAT1, GCAT, GAD1, and NRG1 mRNA expressions were significantly different from those in healthy individuals (all p < 0.05). The ROC analysis of the ΔΔCT values of the target genes for differentiating drug-naïve patients from healthy controls showed an excellent sensitivity (0.960) and modest specificity (0.640) (AUC = 0.889). Drug-free and medicated patients obtained less favorable AUC values while compared to healthy controls. The results for the age- and sex-matched cohort were similar to those of the unmatched cohort. CONCLUSIONS: This is the first study demonstrating that the peripheral mRNA expression levels of NMDAR-related genes may be altered in patients with MDD, especially drug-naïve individuals. The finding supports the NMDAR hypothesis of depression. Whether mRNA expresssion of NMDAR-related genes could serve as a potential biomarker of MDD deserves further investigations.


Assuntos
Transtorno Depressivo Maior/genética , Leucócitos/metabolismo , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Adulto , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Antidepressivos/uso terapêutico , Grupo com Ancestrais do Continente Asiático/genética , Biomarcadores/metabolismo , Estudos de Casos e Controles , Catecol O-Metiltransferase/genética , Catecol O-Metiltransferase/metabolismo , Estudos de Coortes , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/metabolismo , Feminino , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Lipase/genética , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Neuregulina-1/genética , Neuregulina-1/metabolismo , Curva ROC , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Transaminases/genética , Transaminases/metabolismo
19.
Biol Res ; 52(1): 5, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717794

RESUMO

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.


Assuntos
DNA Bacteriano/genética , Rhodobacteraceae/enzimologia , Rhodobacteraceae/isolamento & purificação , Água do Mar/microbiologia , Transaminases/metabolismo , Regiões Antárticas , Técnicas de Tipagem Bacteriana , Filogenia , RNA Ribossômico 16S/genética , Rhodobacteraceae/classificação , Análise de Sequência de DNA
20.
Nat Commun ; 10(1): 15, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604768

RESUMO

In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.


Assuntos
Vias Biossintéticas , Corismato Mutase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Plantas/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexenos/metabolismo , Citosol/metabolismo , Plantas/genética , Plastídeos/genética , Plastídeos/metabolismo , Prefenato Desidratase/genética , Prefenato Desidratase/metabolismo , Transaminases/metabolismo , Sítio de Iniciação de Transcrição , Tirosina/análogos & derivados , Tirosina/metabolismo
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