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1.
J Agric Food Chem ; 68(10): 3195-3202, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32075368

RESUMO

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric ß-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.


Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Galactose/química , Galactosidases/química , Glicosídeo Hidrolases/química , Hexoses/química , Edulcorantes/química , Biocatálise , Flavobacteriaceae/enzimologia , Isomerismo , Lactobacillus sakei/enzimologia
2.
J Clin Pathol ; 73(3): 139-146, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31586937

RESUMO

AIMS: To unveil the role of EI2BL in non-small cell lung cancer (NSCLC) and the relationship between expression of EI2BL and the prognosis of patients with NSCLC. METHODS: Immunohistochemistry (IHC), western blot analysis, immunofluorescence and real-time quantitative PCR (RT-qPCR) were used to evaluate EI2BL protein and mRNA levels in NSCLC and corresponding peritumour tissues. Cell Counting Kit-8, transwell assay and wound healing assay were used to analyse the abilities of cell proliferation, invasion and migration. In addition, the analysis of epithelial-mesenchymal transition (EMT) markers was also assessed by western blot analysis, RT-qPCR and immunofluorescence. Tissue micro-array analysis of 200 NSCLC cases was used to assess the relationship between EI2BL expression and clinicopathological characteristics. Moreover, the prognostic role of EI2BL in 200 patients with NSCLC was evaluated by Cox regression models and Kaplan-Meier methods. RESULTS: Elevated EI2BL expression was more common in NSCLC tissues than paired peritumour tissues in both mRNA and protein level. EI2BL promoted the proliferation, invasion and migration of NSCLC cells. In addition, aberrant EI2BL expression might modulate the expression of key molecules of EMT by ERK1/2 signal pathway. The expression of EI2BL was significantly associated with tumour stage, lymph node metastasis and tumour size. Moreover, higher expression of EI2BL in patients with NSCLC had a poor overall survival rate. CONCLUSIONS: Our study illustrated that elevated expression of EI2BL promoted NSCLC cell proliferation, migration and invasion and EI2BL overexpression may be a reliable biomarker of poor prognosis.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/enzimologia , Aldose-Cetose Isomerases/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Intervalo Livre de Progressão , Transdução de Sinais , Regulação para Cima
3.
Food Chem ; 309: 125710, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31704076

RESUMO

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Clostridiales/enzimologia , Frutose/metabolismo , Aldose-Cetose Isomerases/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Sefarose/química , Temperatura
4.
Enzyme Microb Technol ; 132: 109443, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731969

RESUMO

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Arabinose/metabolismo , Pentoses/metabolismo , Ribose/biossíntese , Aldose-Cetose Isomerases/genética , Alicyclobacillus/enzimologia , Alicyclobacillus/genética , Bacillales/enzimologia , Bacillales/genética , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Pentoses/genética , Temperatura
5.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1040-1050, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793898

RESUMO

In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.


Assuntos
Aldose-Cetose Isomerases/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Insulina Regular de Porco/química , Minerais/química , Modelos Moleculares
6.
PLoS One ; 14(12): e0226010, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31794592

RESUMO

Understanding the regulatory mechanisms that affect obesogenic genes expression in newborns is essential for early prevention efforts, but they remain unclear. Our study aimed to explore whether the maternal p-BMI and GWG were associated with regulatory single-locus DNA methylation in selected obesogenic genes. For this purpose, DNA methylation was assayed by Methylation-Sensitive High Resolution Melting (MS-HRM) technique and Sanger allele-bisulfite sequencing in fifty samples of umbilical vein to evaluate glucosamine-6-phosphate deaminase 2 (GNPDA2), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), and leptin receptor (LEPR) genes. Correlations between DNA methylation levels and indicators of maternal nutritional status were carried out. Western blotting was used to evaluate protein expression in extracts of the same samples. Results indicated that GNPDA2 and PGC1α genes have the same level of DNA methylation in all samples; however, a differential DNA methylation of LEPR gene promoter was found, correlating it with GWG and this correlation is unaffected by maternal age or unhealthy habits. Furthermore, leptin receptor (Lep-Rb) was upregulated in samples that showed the lowest levels of DNA methylation. This study highlights the association between poor GWG and adjustments on obesogenic genes expression in newborn tissues with potential consequences for development of obesity in the future.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Metilação de DNA , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores para Leptina/metabolismo , Veias Umbilicais/metabolismo , Adolescente , Adulto , Aldose-Cetose Isomerases/genética , Índice de Massa Corporal , Feminino , Ganho de Peso na Gestação , Humanos , Estado Nutricional , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Gravidez , Primeiro Trimestre da Gravidez , Regiões Promotoras Genéticas , Receptores para Leptina/genética , Adulto Jovem
7.
Appl Microbiol Biotechnol ; 103(23-24): 9465-9477, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31701197

RESUMO

Discovering sugar metabolism genes is of great interest for lignocellulosic biorefinery. Xylose isomerases (XIs) were commonly screened from metagenomes derived from bovine rumen, soil, and other sources. However, so far, XIs and other sugar-utilizing enzymes have not been discovered from fecal metagenomes. In this study, environmental DNA from the fecal samples collected from yellow cattle (Bos taurus) was sequenced and analyzed. In the whole 14.26 Gbp clean data, 92 putative XIs were annotated. After sequence analysis, seven putative XIs were heterologously expressed in Escherichia coli and characterized in vitro. The XIs 58444 and 58960 purified from E. coli exhibited 22% higher enzyme activity when compared with that of the native E. coli XI. The XI 58444, similar to the XI from Lachnospira multipara, exhibited a relatively stable activity profile across different pH conditions. Four XIs were further investigated in budding yeast Saccharomyces cerevisiae after codon optimization. Overexpression of the codon-optimized 58444 enabled S. cerevisiae to utilize 6.4 g/L xylose after 96 h without any other genetic manipulations, which is 56% higher than the control yeast strain overexpressing an optimized XI gene xylA*3 selected by three rounds of mutation. Our results provide evidence that a bovine fecal metagenome is a novel and valuable source of XIs and other industrial enzymes for biotechnology applications.


Assuntos
Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal , Animais , Biotecnologia , Bovinos , Códon , Escherichia coli/genética , Fezes/microbiologia , Fermentação , Metagenoma , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA
8.
PLoS Pathog ; 15(10): e1008078, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622442

RESUMO

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division.


Assuntos
Antibacterianos/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Fosfomicina/análogos & derivados , Peptidoglicano/biossíntese , Terpenos/metabolismo , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Linhagem Celular Tumoral , Infecções por Chlamydia/patologia , Chlamydia trachomatis/enzimologia , Chlamydia trachomatis/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfomicina/farmacologia , Células HeLa , Humanos
9.
Nat Commun ; 10(1): 4548, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591402

RESUMO

There are many industrially-relevant enzymes that while active, are severely limited by thermodynamic, kinetic, or stability issues (isomerases, lyases, transglycosidases). In this work, we study Lactobacillus sakei L-arabinose isomerase (LsLAI) for D-galactose to D-tagatose isomerization-that is limited by all three reaction parameters. The enzyme demonstrates low catalytic efficiency, low thermostability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we show that encapsulating LsLAI in gram-positive Lactobacillus plantarum that is chemically permeabilized enables reactions at high rates, high conversions, and elevated temperatures. In a batch process, this system enables ~ 50% conversion in 4 h starting with 300 mM galactose (an average productivity of 37 mM h-1), and 85% conversion in 48 h. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Hexoses/metabolismo , Lactobacillus plantarum/enzimologia , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/genética , Biocatálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Isomerismo , Cinética , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Temperatura
10.
Appl Microbiol Biotechnol ; 103(21-22): 8753-8761, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637494

RESUMO

D-Mannose is an epimer of glucose at the C-2 position and exists in nature as a component of mannan. It has 60 and 86% sweetness than that of sucrose and D-glucose, respectively. Because of its low-calorie and nontoxic features, D-mannose is used widely in food, medicine, cosmetic, and food-additive industries. Besides, it exhibits many physiologic benefits on health: immune system, diabetes mellitus, intestinal diseases, and urinary tract infections. It is used as a starting material to synthesize immunostimulatory agents, anti-tumor agents, vitamins, and D-mannitol. However, D-mannose production using chemical synthesis and plant extraction cannot meet the requirements of the industry. This article presents recent research on the biological production of D-mannose. The physiologic benefits and applications of D-mannose are summarized. Besides, different D-mannose-producing enzymes from various sources are discussed in detail with regard to their biochemical characteristics, catalytic efficiency, and reaction kinetics for D-mannose production. Furthermore, attempts to use enzymatic conversion to produce D-mannose are reviewed.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Reatores Biológicos/microbiologia , Manose/metabolismo , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/química , Glucose/química , Plantas/química
11.
Enzyme Microb Technol ; 131: 109427, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615684

RESUMO

d-Ribulose and l-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of d-arabinose and l-fucose byDd-arabinose and l-fucose by d-arabinose isomerase from bacterial sources for the production of d-ribulose and l-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable d-ribulose- and l-fuculose producing d-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The recombinant d-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both d-arabinose and l-fucose were observed to be 98.08 and 85.52 U mg-1, respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn2+ was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both d-arabinose and l-fucose at 75 °C after 10 h. The Michaelis-Menten constant (Km), the turnover number (kcat) and catalytic efficiency (kcat/Km) for d-arabinose/l-fucose were 111/81.24 mM, 18,466/10,688 min-1, and 166/132 mM-1  min-1, respectively. When the reaction reached to equilibrium, the conversion rates of d-ribulose from d-arabinose and l-fuculose from l-fucose were almost 27% (21 g L-1) and 24.88% (19.92 g L-1) from 80 g L-1 of d-arabinose and l-fucose, respectively.


Assuntos
Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Arabinose/metabolismo , Firmicutes/enzimologia , Hexoses/metabolismo , Pentoses/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/isolamento & purificação , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
12.
Nat Methods ; 16(10): 979-982, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527838

RESUMO

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.


Assuntos
Cristalografia/métodos , Síncrotrons , Acetilglucosamina/química , Aldose-Cetose Isomerases/química , Glucose/química , Muramidase/química , Estudo de Prova de Conceito
13.
Biotechnol J ; 14(11): e1900114, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31294913

RESUMO

Xylose/glucose isomerases are important industrial enzymes that are most widely used in food industries; however, their previously reported expression levels do not meet the requirements for industrial application. Here, an antibiotic resistance marker (ARM)-free system driven by ribosomal RNA (rRNA) promoters is developed to obtain high-level xylose/glucose isomerase (XI/GI) expression in Streptomyces rubiginosus (S. rubiginosus). The rRNA promoter rrnD yields the highest glucose isomerase production titer of XIs/GIs, which is eight times higher than that of ermEp* and 2.6 times higher than that of kasOp*. The integrated ARM gene is removed by further introduction of the Cre plasmid with a temperature-sensitive replicon. The production titer of XIs/GIs is further improved by replacing the xylR gene with an additional expression glucose isomerase cassette at the xylR locus. Ultimately, the glucose isomerase activity reaches up to 79.7 ± 7.5 U mL-1 at 96 h. The results support the robustness and stability of XI/GI production with this ARM-free system using optimal ribosomal promoters in S. rubiginosus, demonstrating strong potential in large-scale industrial applications. Besides, the results imply that rRNA promoters are strong promoters that can be used for protein engineering or metabolic engineering.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Glucose/metabolismo , Regiões Promotoras Genéticas/genética , Ribossomos/genética , Streptomyces/genética , Streptomyces/metabolismo , Xilose/metabolismo , Dissacarídeos , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Engenharia Metabólica , Plasmídeos , Análise de Sequência
14.
J Synchrotron Radiat ; 26(Pt 4): 931-944, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31274415

RESUMO

Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13-3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.


Assuntos
Aldose-Cetose Isomerases/química , Cristalografia por Raios X/métodos , Raios X , Aminoácidos/química , Modelos Moleculares , Conformação Proteica
15.
Plant Physiol Biochem ; 142: 94-105, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279136

RESUMO

1-Deoxy-D-xylulose-5-phosphate synthasse (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) are key enzymes in terpenoid biosynthesis. DXS catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde-3-phosphate. DXR catalyzes the formation of 2-C-methyl-D-erythritol 4-phosphate (MEP) from DXP. Previous studies of the DXS and DXR genes have focused on herbs, such as Arabidopsis thaliana, Salvia miltiorrhiza, and Amomum villosum, but few studies have been conducted on woody plants. For that reason, we chose Populus trichocarpa as a model woody plant for investigating the DXS and DXR genes. PtDXS exhibited the highest expression level in leaves and the lowest expression in roots. PtDXR showed maximum expression in young leaves, and the lowest expression in mature leaves. The expression profiles revealed by RT-PCR following different elicitor treatments such as abscisic acid, NaCl, PEG6000, H2O2, and cold stress showed that PtDXS and PtDXR were elicitor-responsive genes. Our results showed that the PtDXS gene exhibited diurnal changes, but PtDXR did not. Moreover, overexpression of PtDXR in transgenic poplars improved tolerance to abiotic and biotic stresses. Those results showed that the PtDXR encoded a functional protein, and widely participates in plant growth and development, stress physiological process.


Assuntos
Aldose-Cetose Isomerases/genética , Proteínas de Plantas/genética , Populus/genética , Transferases/genética , Ácido Abscísico/farmacologia , Aldose-Cetose Isomerases/metabolismo , Ritmo Circadiano , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/farmacologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/efeitos dos fármacos , Populus/fisiologia , Saccharomycetales/patogenicidade , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Transferases/metabolismo
16.
Molecules ; 24(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295807

RESUMO

The rate-limiting enzyme of the 2-methyl-d-erythritol-4-phosphate (MEP) terpenoid biosynthetic pathway, 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), provides the perfect target for screening new antibacterial substances. In this study, we tested the DXR inhibitory effect of 35 plant essential oils (EOs), which have long been recognized for their antimicrobial properties. The results show that the EOs of Zanbthoxylum bungeanum (ZB), Schizonepetae tenuifoliae (ST), Thymus quinquecostatus (TQ), Origanum vulgare (OV), and Eugenia caryophyllata (EC) displayed weak to medium inhibitory activity against DXR, with IC50 values of 78 µg/mL, 65 µg/mL, 59 µg/mL, 48 µg/mL, and 37 µg/mL, respectively. GC-MS analyses of the above oils and further DXR inhibitory activity tests of their major components revealed that eugenol (EC) and carvacrol (TQ and OV) possess medium inhibition against the protein (68.3% and 55.6%, respectively, at a concentration of 20 µg/mL), whereas thymol (ST, TQ, and OV), carveol (ZB), and linalool (ZB, ST, and OV) only exhibited weak inhibition against DXR, at 20 µg/mL (23%-26%). The results add more details to the antimicrobial mechanisms of plant EOs, which could be very helpful in the direction of the reasonable use of EOs in the food industry and in the control of phytopathogenic microbials.


Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Antibacterianos/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Óleos Voláteis/química , Fotometria/métodos , Óleos Vegetais/química
17.
Plant Physiol Biochem ; 142: 43-52, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31272034

RESUMO

Yarrow (Achillea millefolium) is a medicinal plant from the Asteracea which biosynthesize different secondary metabolites especially terpenes and phenylpropanoids. To improve our understanding of the regulatory mechanisms behind the biosynthesis of these compounds we analyzed the expression of some genes associated with the biosynthesis of terpenes and phenylpropanoids in different tissues and in response to trans-cinnamic acid (tCA) as an inhibitor of PAL activity. Isolation and expression analysis of DXR, GPPS, PAL and CHS genes together with linalool synthase (LIS) as monoterpene synthase was conducted in different developmental stages of leaves, flowers and in response to trans-cinnamic acid (tCA). Differential expression of these genes observed in different tissues. tCA up-regulated the biosynthetic genes of monterpenes and down-regulated the biosynthetic genes of phenylpropanoids. Gene expression analysis in intact leaves and leaves without glandular trichomes showed that DXR, LIS, PAL and CHS are highly expressed in glandular trichomes while GPPS expressed ubiquitously. Analysis of essential oils composition showed that sesquiterpenes and monoterpenes are main compounds; in which from 57 identified compounds the highest were germacreneD (% 11.5), guaiol (%10.38), spatulenol (%8.73) and caryophyllene oxide (%7.48).


Assuntos
Achillea/genética , Achillea/metabolismo , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Terpenos/metabolismo , Achillea/química , Achillea/efeitos dos fármacos , Aciltransferases/genética , Aciltransferases/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Vias Biossintéticas , Cinamatos/farmacologia , Farnesiltranstransferase/genética , Farnesiltranstransferase/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Hidroliases/genética , Hidroliases/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Tricomas/genética , Tricomas/metabolismo
18.
Eur J Med Genet ; 62(8): 103708, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31247379

RESUMO

Ribose 5-phosphate isomerase deficiency is a rare genetic leukoencephalopathy caused by pathogenic sequence variants in RPIA, that encodes ribose 5-phosphate isomerase, an enzyme in the pentose phosphate pathway. Till date, only three individuals with ribose 5-phosphate isomerase deficiency have been described in literature. We report on a subject with RPIA associated progressive leukoencephalopathy with elevated urine arabitol and ribitol levels and a novel missense variant c.770T > C p.(Ile257Thr) in exon 8 of RPIA. We also compare the phenotypes of all the four subjects. Our report confirms the phenotype and the genetic cause of this condition.


Assuntos
Aldose-Cetose Isomerases/deficiência , Erros Inatos do Metabolismo dos Carboidratos/genética , Leucoencefalopatias/genética , Polineuropatias/genética , Aldose-Cetose Isomerases/genética , Alelos , Erros Inatos do Metabolismo dos Carboidratos/tratamento farmacológico , Erros Inatos do Metabolismo dos Carboidratos/patologia , Humanos , Leucoencefalopatias/tratamento farmacológico , Leucoencefalopatias/patologia , Masculino , Via de Pentose Fosfato/genética , Polineuropatias/tratamento farmacológico , Polineuropatias/patologia , Ribitol/administração & dosagem , Álcoois Açúcares/administração & dosagem
19.
Rev Sci Instrum ; 90(5): 054101, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31153273

RESUMO

We have developed a handling-free mounting method for X-ray crystallography of protein crystals at room temperatures-the glass capillary method. In this method, crystals were nucleated and grown on the capillary walls, and then, growth solutions were gently removed. The procedures for collecting data on the crystals were conducted by simply setting the capillary on the goniometer of a synchrotron beamline without touching the crystals. Crystal quality was characterized using mosaicity, resolution at I/σ(I) = 2, I/σ(I) at resolution = 2.0 Å, Rmerge, and completeness. Wilson plots were also used to characterize the quality of crystals. In particular, all samples showed very low mosaicity; the handling-free method successfully retained their low mosaicity and effectively maintained the crystal quality.


Assuntos
Aldose-Cetose Isomerases/química , Cristalografia por Raios X/instrumentação , Síncrotrons , Temperatura
20.
Metab Eng ; 55: 1-11, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31150803

RESUMO

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae, i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae , Xilose/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Xilose/genética
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