Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 272
Filtrar
1.
Science ; 383(6683): eadg9196, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38330117

RESUMO

Plants measure daylength (photoperiod) to regulate seasonal growth and flowering. Photoperiodic flowering has been well studied, but less is known about photoperiodic growth. By using a mutant with defects in photoperiodic growth, we identified a seasonal growth regulation pathway that functions in long days in parallel to the canonical long-day photoperiod flowering mechanism. This is achieved by using distinct mechanisms to detect different photoperiods: The flowering pathway measures photoperiod as the duration of light intensity, whereas the growth pathway measures photoperiod as the duration of photosynthetic activity (photosynthetic period). Plants can then independently control expression of genes required for flowering or growth. This demonstrates that seasonal flowering and growth are dissociable, allowing them to be coordinated independently across seasons.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ritmo Circadiano , Flores , Mio-Inositol-1-Fosfato Sintase , Fotoperíodo , Ritmo Circadiano/genética , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Estações do Ano , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia
2.
Methods Enzymol ; 685: 57-93, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37245915

RESUMO

Phosphate ester analogs in which the bridging oxygen is replaced with a methylene or fluoromethylene group are well known non-hydrolyzable mimics of use as inhibitors and substrate analogs for reactions involving phosphate esters. Properties of the replaced oxygen are often best mimicked by a mono-fluoromethylene group, but such groups are challenging to synthesize and can exist as two stereoisomers. Here, we describe the protocol for our method of synthesizing the α-fluoromethylene analogs of d-glucose 6-phosphate (G6P), as well as the methylene and difluoromethylene analogs, and their application in the study of 1l-myo-inositol-1-phosphate synthase (mIPS). mIPS catalyzes the synthesis of 1l-myo-inositol 1-phosphate (mI1P) from G6P, in an NAD-dependent aldol cyclization. Its key role in myo-inositol metabolism makes it a putative target for the treatment of several health disorders. The design of these inhibitors allowed for the possibility of substrate-like behavior, reversible inhibition, or mechanism-based inactivation. In this chapter, the synthesis of these compounds, expression and purification of recombinant hexahistidine-tagged mIPS, the mIPS kinetic assay and methods for determining the behavior of the phosphate analogs in the presence of mIPS, and a docking approach to rationalizing the observed behavior are described.


Assuntos
Glucose-6-Fosfato , Organofosfonatos , Mio-Inositol-1-Fosfato Sintase/química , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fosfatos , Glucose
3.
Protoplasma ; 260(4): 1097-1107, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36602620

RESUMO

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) encodes the enzyme synthesizing Myo-inositol for plant growth and development. Myo-inositol and its phosphate derivatives are involved in various physiological functions ranging from cell wall synthesis, chromatin remodeling, signal transduction, and providing stress responses. In the present study, we report that MIPS regulates chlorophyll content and photosynthesis efficiency via the ethylene signaling pathway. We have used Triticum aestivum MIPS-A (TAMIPS-A) for the present study and characterized it by mutant complementation and overexpression studies in Arabidopsis. TaMIPS-A overexpressing Arabidopsis transgenics were analyzed physiologically under thermal stress conditions. Analysis of overexpression TaMIPS-A transgenics under control and thermal stress conditions revealed them to have enhanced photosynthetic potential under heat stress. When TaMIPS-A overexpression (OE) Arabidopsis transgenics are supplemented with either ACC, the ethylene precursor, or AgNO3, the ethylene signaling inhibitor indicated that MIPS regulates the photosynthetic efficiency and chlorophyll content via the ethylene signaling pathway under control and thermal stress. Expression analysis of essential genes involved in the ethylene biosynthetic and signaling pathway corroborated.


Assuntos
Arabidopsis , Fosfatos de Inositol , Arabidopsis/metabolismo , Clorofila , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fosfatos , Fotossíntese , Etilenos , Resposta ao Choque Térmico
4.
Biochemistry ; 61(10): 868-878, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35467843

RESUMO

The biosynthesis of myo-inositol (mI) is central to the function of many organisms across all kingdoms of life. The first and rate-limiting step in this pathway is catalyzed by 1l-myo-inositol 1-phosphate synthase (mIPS), which converts d-glucose 6-phosphate (G6P) into 1l-myo-inositol 1-phosphate (mI1P). Extensive studies have shown that this reaction occurs through a stepwise NAD+-dependent redox aldol cyclization mechanism producing enantiomerically pure mI1P. Although the stereochemical nature of the mechanism has been elucidated, there is a lack of understanding of the importance of amino acid residues in the active site. Crystal structures of mIPS in the ternary complex with substrate analogues and NAD(H) show different ligand orientations. We therefore proposed to use isosteric and isoelectronic analogues of G6P to probe the active site. Here, we report the synthesis of the methylenephosphonate, difluoromethylenephosphonate, and (R)- and (S)-monofluoromethylenephosphonate analogues of G6P and their evaluation as inhibitors of mIPS activity. While the CH2 and CF2 analogues were produced with slight modification of a previously described route, the CHF analogues were synthesized through a new, shorter pathway. Kinetic behavior shows that all compounds are reversible competitive inhibitors with respect to G6P, with Ki values in the order CF2 (0.18 mM) < (S)-CHF (0.24 mM) < (R)-CHF (0.59 mM) < CH2 (1.2 mM). Docking studies of these phosphonates using published crystal structures show that substitution of the oxygen atom of the substrate changes the conformation of the resulting inhibitors, altering the position of carbon-6 and carbon-5, and this change is more pronounced with fluorine substitution.


Assuntos
Mio-Inositol-1-Fosfato Sintase , Organofosfonatos , Carbono , Domínio Catalítico , Glucose , Glucose-6-Fosfato , Fosfatos de Inositol , Mio-Inositol-1-Fosfato Sintase/química , NAD/metabolismo , Organofosfonatos/química , Fosfatos
5.
J Biomol Struct Dyn ; 40(8): 3371-3384, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-33200690

RESUMO

Myo-inositol is one of the vital nutritional requirements for the Leishmania parasites' survival and virulence in the mammalian host. . Myo-inositol-1-phosphate synthase (MIPS) is responsible for the synthesis of myo-inositol in Leishmania, which plays a vital role in Leishmania's virulence to mammalian hosts. Earlier studies suggest MIP synthase as a potential drug target against which valproate was used as a drug. So, MIP synthase can be used as a target for anti-leishmanial drugs, and its inhibition may help in preventing leishmaniasis. The present study aims to identify valproate's potent analogs as drugs against MIP synthase of L. donovani (Ld-MIPS) with minimum side effects and toxicity to host.In this study, the three-dimensional structure of Ld-MIPS was built, followed by active site prediction. Ligand-based virtual screening was done using hybrid similarity recognition methods. The best 123 valproate analogs were filtered based on their quantitative structure activity relationship (QSAR) properties and were docked against Ld-MIPS using FlexX, PyRx and iGEMDOCK software. The topmost five ligands were selected for molecular dynamics simulation and pharmacokinetic analysis based on the docking score. Simulation studies up to 30 ns revealed that all five lead molecules bound with Ld-MIPS throughout MD simulation and there was no variation in their backbone. All the chosen inhibitors exhibited good pharmacokinetics/ADMET predictions with an excellent absorption profile, metabolism, oral bioavailability, solubility, excretion, and minimal toxicity, suggesting that these inhibitors may further be developed as anti-leishmaniasis drugs to prevent the spread of leishmaniasis.Communicated by Ramaswamy H. Sarma.


Assuntos
Leishmania donovani , Leishmaniose , Animais , Inositol/farmacologia , Ligantes , Mamíferos , Simulação de Dinâmica Molecular , Mio-Inositol-1-Fosfato Sintase , Ácido Valproico/farmacologia
6.
Microb Cell Fact ; 20(1): 138, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281557

RESUMO

BACKGROUND: Myo-Inositol Phosphate Synthase (MIP) catalyzes the conversion of glucose 6- phosphate into inositol phosphate, an essential nutrient and cell signaling molecule. Data obtained, first in bovine brain and later in plants, established MIP expression in organelles and in extracellular environments. A physiological role for secreted MIP has remained elusive since its first detection in intercellular space. To provide further insight into the role of MIP in intercellular milieus, we tested the hypothesis that MIP may function as a growth factor, synthesizing inositol phosphate in intercellular locations requiring, but lacking ability to produce or transport adequate quantities of the cell-cell communicator. This idea was experimentally challenged, utilizing a Saccharomyces cerevisiae inositol auxotroph with no MIP enzyme, permeable membranes with a 0.4 µm pore size, and cellular supernatants as external sources of inositol isolated from S. cerevisiae cells containing either wild-type enzyme (Wt-MIP), no MIP enzyme, auxotroph (Aux), or a green fluorescent protein (GFP) tagged reporter enzyme (MIP- GFP) in co- culturing experiments. RESULTS: Resulting cell densities and microscopic studies with corroborating biochemical and molecular analyses, documented sustained growth of Aux cells in cellular supernatant, concomitant with the uptakeof MIP, detected as MIP-GFP reporter enzyme. These findings revealed previously unknown functions, suggesting that the enzyme can: (1) move into and out of intercellular space, (2) traverse cell walls, and (3) act as a growth factor to promote cellular proliferation of an inositol requiring cell. CONCLUSIONS: Co-culturing experiments, designed to test a probable function for MIP secreted in extracellular vesicles, uncovered previously unknown functions for the enzyme and advanced current knowledge concerning spatial control of inositol phosphate biosynthesis. Most importantly, resulting data identified an extracellular vesicle (a non-viral vector) that is capable of synthesizing and transporting inositol phosphate, a biological activity that can be used to enhance specificity of current inositol phosphate therapeutics.


Assuntos
Fosfatos de Inositol/metabolismo , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Fosfatos de Inositol/biossíntese , Técnicas Microbiológicas/métodos , Mio-Inositol-1-Fosfato Sintase/genética , Saccharomyces cerevisiae/genética
7.
Plant Sci ; 301: 110654, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33218625

RESUMO

Myo-inositol and its derivatives play vital roles in plant stress tolerance. Myo-inositol-1-phosphate synthase (MIPS) is the rate-limiting enzyme of myo-inositol biosynthesis. However, the role of apple MIPS-mediated myo-inositol biosynthesis in stress tolerance remains elusive. In this study, we found that ectopic expression of MdMIPS1 from apple increased myo-inositol content and enhanced tolerance to salt and osmotic stresses in transgenic Arabidopsis lines. In transgenic apple lines over-expressing MdMIPS1, the increased myo-inositol levels could promote accumulation of other osmoprotectants such as glucose, sucrose, galactose, and fructose, to alleviate salinity-induced osmotic stress. Also, it was shown that overexpression of MdMIPS1 enhanced salinity tolerance by improving the antioxidant system to scavenge ROS, as well as Na+ and K+ homeostasis. Taken together, our results revealed a protective role of MdMIPS1-mediated myo-inositol biosynthesis in salt tolerance by improving osmotic balance, antioxidant defense system, and ion homeostasis in apple.


Assuntos
Antioxidantes/metabolismo , Malus/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Tolerância ao Sal/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inositol/metabolismo , Íons/metabolismo , Malus/fisiologia , Mio-Inositol-1-Fosfato Sintase/genética , Osmose , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Salinidade , Estresse Fisiológico
8.
Sci Rep ; 10(1): 17329, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060662

RESUMO

Myo-inositol is a ubiquitous metabolite of plants. It is synthesized by a highly conserved enzyme L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4). Myo-inositol is well characterized during abiotic stress tolerance but its role during growth and development is unclear. In this study, we demonstrate that the apical hook maintenance and hypocotyl growth depend on myo-inositol. We discovered the myo-inositol role during hook formation and its maintenance via ethylene pathway in Arabidopsis by supplementation assays and qPCR. Our results suggest an essential requirement of myo-inositol for mediating the ethylene response and its interaction with brassinosteroid to regulate the skotomorphogenesis. A model is proposed outlining how MIPS regulates apical hook formation and hypocotyl growth.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Inositol/metabolismo , Morfogênese , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genes de Plantas , Hipocótilo/crescimento & desenvolvimento , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Estresse Fisiológico
9.
Sci Rep ; 10(1): 10766, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612188

RESUMO

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) is involved in abiotic stress tolerance, however its disruption and overexpression has also been associated with enhanced tolerance to pathogens. The molecular mechanism underlying the role of MIPS in growth, immunity and abiotic stress tolerance remains uncharacterized. We explore the molecular mechanism of MIPS action during growth and heat stress conditions. We raised and characterized the TaMIPS over-expressing rice transgenics which showed a reduced reproductive potential. Transcriptome analysis of overexpression transgenics revealed the activation of ET/JA dependent immune response. Pull-down analysis revealed the interaction of TaMIPS-B with ethylene related proteins. Our results suggest an essential requirement of MIPS for mediating the ethylene response and regulate the growth. A model is proposed outlining how fine tuning of MIPS regulate growth and stress tolerance of the plant.


Assuntos
Etilenos/química , Inositol/química , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fosfatos/química , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , Mineração de Dados , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Oryza/genética , Fenótipo , Fotossíntese , Plantas Geneticamente Modificadas , Sementes/metabolismo , Transdução de Sinais , Estresse Fisiológico , Transcriptoma , Transgenes , Triticum/genética
10.
Lipids ; 55(5): 469-477, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32542681

RESUMO

Soybean (Glycine max) meal is an important protein source. Soybean meal with lower phytate and oligosaccharides improves meal quality. A single recessive mutation in soybean myo-inositol 1-phosphate synthase (Gm-lpa-TW75-1) confers a seed phenotype with low phytate and increased inorganic phosphate. The mutant was crossed with high oil lines expressing a diacylglycerol acyltransferase1 (DGAT) gene from Vernonia galamensis (VgD). Gm-lpa-TW75-1 X VgD, designated GV, has 21%, and 22% oil and 41% and 43% protein from field and greenhouse seed production, respectively. No significant differences were found in mineral concentrations except for Fe which was 229 µg/g dry mass for GV followed by 174.3 for VgD and 162 for Gm-lpa-TW75-1. Phosphate (Pi) is higher in Gm-lpa-TW75-1 as expected at 5 mg/g, followed by GV at 1.6 mg/g whereas Jack, VgD, and Taiwan75 have about 0.3 mg/g. The Gm-lpa-TW75-1 line has the lowest phytate concentration at 1.4 mg/g followed by GV with 1.8 mg/g compared to Taiwan75, VgD, and Jack with 2.5 mg/g. This work describes a high oil and protein soybean line, GV, with increased Pi and lower phytate which will increase the nutritional value for human and animal feed.


Assuntos
Diacilglicerol O-Aciltransferase/genética , Mio-Inositol-1-Fosfato Sintase/genética , Plantas Geneticamente Modificadas/genética , Técnicas de Inativação de Genes , Fosfatos de Inositol/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , /crescimento & desenvolvimento , Vernonia/enzimologia , Vernonia/genética
11.
PLoS One ; 15(3): e0230572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210477

RESUMO

Chromatin structure plays a decisive role in gene regulation through the actions of transcriptional activators, coactivators, and epigenetic machinery. These trans-acting factors contribute to gene expression through their interactions with chromatin structure. In yeast INO1 activation, transcriptional activators and coactivators have been defined through intense study but the mechanistic links within these trans-acting factors and their functional implications are not yet fully understood. In this study, we examined the crosstalk within transcriptional coactivators with regard to the implications of Snf2p acetylation during INO1 activation. Through various biochemical analysis, we demonstrated that both Snf2p and Ino80p chromatin remodelers accumulate at the INO1 promoter in the absence of Snf2p acetylation during induction. Furthermore, nucleosome density and histone acetylation patterns remained unaffected by Snf2p acetylation status. We also showed that cells experience increased sensitivity to copper toxicity when remodelers accumulate at the INO1 promoter due to the decreased CUP1 expression. Therefore, our data provide evidence for crosstalk within transcriptional co-activators during INO1 activation. In light of these findings, we propose a model in which acetylation-driven chromatin remodeler recycling allows for efficient regulation of genes that are dependent upon limited co-activators.


Assuntos
Adenosina Trifosfatases/metabolismo , Metalotioneína/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Cobre/metabolismo , Cobre/toxicidade , Histonas/metabolismo , Metalotioneína/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Ativação Transcricional
12.
Commun Biol ; 3(1): 93, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123276

RESUMO

A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.


Assuntos
Bacillus subtilis/metabolismo , Glucose/metabolismo , Inositol/biossíntese , Engenharia Metabólica/métodos , Doença de Alzheimer/tratamento farmacológico , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Inativação Gênica , Humanos , Inositol/uso terapêutico , Mycobacterium tuberculosis/genética , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Organismos Geneticamente Modificados
13.
Dev Cell ; 52(3): 309-320.e5, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31902656

RESUMO

Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.


Assuntos
Montagem e Desmontagem da Cromatina , Cromossomos Fúngicos , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Histonas/genética , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
14.
Biochemistry ; 58(51): 5112-5116, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31825604

RESUMO

The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.


Assuntos
Adenosina/análogos & derivados , Mio-Inositol-1-Fosfato Sintase/metabolismo , Adenosina/biossíntese , Adenosina/química , Modelos Moleculares , Mio-Inositol-1-Fosfato Sintase/química , Conformação Proteica , Estereoisomerismo , Streptomyces/enzimologia
15.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096655

RESUMO

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.


Assuntos
/genética , Inositol/análogos & derivados , Água/metabolismo , Secas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Inositol/biossíntese , Inositol/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Estresse Fisiológico , Sacarose/metabolismo , Transcriptoma
16.
Sci Rep ; 9(1): 7744, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123331

RESUMO

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.


Assuntos
/genética , Mio-Inositol-1-Fosfato Sintase/genética , Ácido Fítico/metabolismo , Disponibilidade Biológica , Fabaceae/genética , Fabaceae/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Engenharia Genética/métodos , Minerais/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fósforo/metabolismo , Ácido Fítico/efeitos adversos , Ácido Fítico/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , RNA Antissenso/genética , Proteínas de Armazenamento de Sementes/genética , Sementes/genética , /crescimento & desenvolvimento
17.
J Agric Food Chem ; 67(17): 5043-5052, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30977368

RESUMO

The low phytic acid ( lpa) soybean ( Glycine max L. Merr.) mutant Gm-lpa-TW-1-M, resulting from a 2 bp deletion in GmMIPS1, was crossed with a commercial cultivar. F3 and F5 progenies were subjected to nontargeted GC-based metabolite profiling, allowing analysis of a broad array of low molecular weight constituents. In the homozygous lpa mutant progenies the intended phytic acid reduction was accompanied by remarkable metabolic changes of nutritionally relevant constituents such as reduced contents of raffinose oligosaccharides and galactosyl cyclitols as well as increased concentrations in sucrose and various free amino acids. The mutation-induced metabolite signature was nearly unaffected by the cross-breeding and consistently expressed over generations and in different growing seasons. Therefore, not only the primary MIPS1 lpa mutant but also its progenies might be valuable genetic resources for commercial breeding programs to produce soybean seeds stably exhibiting improved phytate-related and nutritional properties.


Assuntos
Proteínas de Arabidopsis/genética , Mio-Inositol-1-Fosfato Sintase/genética , Ácido Fítico/análise , Proteínas de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Homozigoto , Hibridização Genética , Mutação , Mio-Inositol-1-Fosfato Sintase/metabolismo , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Ácido Fítico/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Rafinose/análise , Rafinose/metabolismo , /genética , Sacarose/análise , Sacarose/metabolismo
18.
Int J Mol Sci ; 20(5)2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30862084

RESUMO

Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes de Plantas , Gossypium/genética , Mio-Inositol-1-Fosfato Sintase/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Expressão Ectópica do Gene , Éxons , Regulação da Expressão Gênica de Plantas , Íntrons , Mutação com Perda de Função , Família Multigênica , Mio-Inositol-1-Fosfato Sintase/química , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fenótipo , Filogenia
19.
Genomics ; 111(6): 1929-1945, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30660787

RESUMO

The myo-inositol biosynthesis pathway triggering protein MIPS is best known for its necessity, ubiquitous nature and occurrence throughout all living kingdom. However, the functional disparity of MIPS genes in green plant is still viable. The present work considered a comprehensive genome-wide analysis from sequenced plants to identify MIPS homologs in respective organisms and their genomic architecture. Variation of MIPS gene expression in twelve different species in diverse conditions has also been analysed. All MIPS genes share a conserved sequence property in most of its coding region, but its regulatory elements, gene structure and expression network vary significantly. Phylogenetic inference confirms the evolution of MIPS from a single common algal ancestor to seed plants and acquiring functional variation through genomic control. This paper represents MIPS as a model for studying gene duplication, functional divergence and diversification events in plant lineages.


Assuntos
Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência Conservada , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Inositol/metabolismo , Família Multigênica , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Plantas/metabolismo , Sequências Reguladoras de Ácido Nucleico
20.
Microbiologyopen ; 8(5): e00721, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30270521

RESUMO

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.


Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/fisiologia , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Aerobiose , Deleção de Genes , Viabilidade Microbiana , Mio-Inositol-1-Fosfato Sintase/genética , Carbonilação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...