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1.
Plant Biotechnol J ; 20(8): 1606-1621, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35514029

RESUMO

Genetically enhancing drought tolerance and nutrient use efficacy enables sustainable and stable wheat production in drought-prone areas exposed to water shortages and low soil fertility, due to global warming and declining natural resources. In this study, wheat plants, exhibiting improved drought tolerance and N-use efficacy, were developed by introducing GmTDN1, a gene encoding a DREB-like transcription factor, into two modern winter wheat varieties, cv Shi4185 and Jimai22. Overexpressing GmTDN1 in wheat resulted in significantly improved drought and low-N tolerance under drought and N-deficient conditions in the greenhouse. Field trials conducted at three different locations over a period of 2-3 consecutive years showed that both Shi4185 and Jimai22 GmTDN1 transgenic lines were agronomically superior to wild-type plants, and produced significantly higher yields under both drought and N-deficient conditions. No yield penalties were observed in these transgenic lines under normal well irrigation conditions. Overexpressing GmTDN1 enhanced photosynthetic and osmotic adjustment capacity, antioxidant metabolism, and root mass of wheat plants, compared to those of wild-type plants, by orchestrating the expression of a set of drought stress-related genes as well as the nitrate transporter, NRT2.5. Furthermore, transgenic wheat with overexpressed NRT2.5 can improve drought tolerance and nitrogen (N) absorption, suggesting that improving N absorption in GmTDN1 transgenic wheat may contribute to drought tolerance. These findings may lead to the development of new methodologies with the capacity to simultaneously improve drought tolerance and N-use efficacy in cereal crops to ensure sustainable agriculture and global food security.


Assuntos
Secas , Triticum , Regulação da Expressão Gênica de Plantas , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Triticum/genética , Triticum/metabolismo
2.
Front Genet ; 12: 784545, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34966414

RESUMO

Wheat is one of the most important food crops in the world, with development of the grains directly determining yield and quality. Understanding grain development and the underlying regulatory mechanisms is therefore essential in improving the yield and quality of wheat. In this study, the developmental characteristics of the pericarp was examined in developing wheat grains of the new variety Jimai 70. As a result, pericarp thickness was found to be thinnest in grains at the top of the spike, followed by those in the middle and thickest at the bottom. Moreover, this difference corresponded to the number of cell layers in the pericarp, which decreased as a result of programmed cell death (PCD). A number of autophagy-related genes (ATGs) are involved in the process of PCD in the pericarp, and in this study, an increase in ATG8-PE expression was observed followed by the appearance of autophagy structures. Meanwhile, following interference of the key autophagy gene ATG8, PCD was inhibited and the thickness of the pericarp increased, resulting in small premature grains. These findings suggest that autophagy and PCD coexist in the pericarp during early development of wheat grains, with both processes increasing from the bottom to the top of the spike. Moreover, PCD was also found to rely on ATG8-mediated autophagy. The results of this study therefore provide a theoretical basis for in-depth studies of the regulatory mechanisms of wheat grain development.

3.
Int J Mol Sci ; 20(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744172

RESUMO

Although studies have shown the concomitant occurrence of autophagic and programmed cell death (PCD) in plants, the relationship between autophagy and PCD and the factors determining this relationship remain unclear. In this study, seedlings of the wheat cultivar Jimai 22 were used to examine the occurrence of autophagy and PCD during polyethylene glycol (PEG)-8000-induced drought stress. Autophagy and PCD occurred sequentially, with autophagy at a relatively early stage and PCD at a much later stage. These findings suggest that the duration of drought stress determines the occurrence of PCD following autophagy. Furthermore, the addition of 3-methyladenine (3-MA, an autophagy inhibitor) and the knockdown of autophagy-related gene 6 (ATG6) accelerated PEG-8000-induced PCD, respectively, suggesting that inhibition of autophagy also results in PCD under drought stress. Overall, these findings confirm that wheat seedlings undergo autophagic survival under mild drought stress, with subsequent PCD only under severe drought.


Assuntos
Apoptose , Autofagia , Secas , Triticum/crescimento & desenvolvimento , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Polietilenoglicóis/toxicidade , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Triticum/metabolismo
4.
Front Genet ; 9: 12, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29441089

RESUMO

The 14-3-3 gene family members play key roles in various cellular processes. However, little is known about the numbers and roles of 14-3-3 genes in wheat. The aims of this study were to identify TaGF14 numbers in wheat by searching its whole genome through blast, to study the phylogenetic relationships with other plant species and to discuss the functions of TaGF14s. The results showed that common wheat harbored 20 TaGF14 genes, located on wheat chromosome groups 2, 3, 4, and 7. Out of them, eighteen TaGF14s are non-ε proteins, and two wheat TaGF14 genes, TaGF14i and TaGF14f, are ε proteins. Phylogenetic analysis indicated that these genes were divided into six clusters: cluster 1 (TaGF14d, TaGF14g, TaGF14j, TaGF14h, TaGF14c, and TaGF14n); cluster 2 (TaGF14k); cluster 3 (TaGF14b, TaGF14l, TaGF14m, and TaGF14s); cluster 4 (TaGF14a, TaGF14e, and TaGF14r); cluster 5 (TaGF14i and TaGF14f); and cluster 6 (TaGF14o, TaGF14p, TaGF14q, and TaGF14t). Tissue-specific gene expressions suggested that all TaGF14s were likely constitutively expressed, except two genes, i.e., TaGF14p and TaGF14f. And the highest amount of TaGF14 transcripts were observed in developing grains at 20 days post anthesis (DPA), especially for TaGF14j and TaGF14l. After drought stress, five genes, i.e., TaGF14c, TaGF14d, TaGF14g, TaGF14h, and TaGF14j, were up-regulated expression under drought stress for both 1 and 6 h, suggesting these genes played vital role in combating against drought stress. However, all the TaGF14s were down-regulated expression under heat stress for both 1 and 6 h, indicating TaGF14s may be negatively associated with heat stress by reducing the expression to combat heat stress or through other pathways. These results suggested that cluster 1, e.g., TaGF14j, may participate in the whole wheat developing stages, e.g., grain-filling (starch biosynthesis) and may also participate in combating against drought stress. Subsequently, a homolog of TaGF14j, TaGF14-JM22, were cloned by RACE and used to validate its function. Immunoblotting results showed that TaGF14-JM22 protein, closely related to TaGF14d, TaGF14g, and TaGF14j, can interact with AGP-L, SSI, SSII, SBEIIa, and SBEIIb in developing grains, suggesting that TaGF14s located on group 4 may be involved in starch biosynthesis. Therefore, it is possible to develop starch-rich wheat cultivars by modifying TaGF14s.

5.
Cytogenet Genome Res ; 147(2-3): 186-94, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26836300

RESUMO

Aegilops mutica Boiss., a diploid species (2n = 2x = 14, TT), has been rarely studied before. In this research, a hexaploid wheat (cv. Chinese Spring)-Ae. mutica partial amphiploid and a wheat-Ae. mutica addition line were characterized by chromosome karyotyping, FISH using oligonucleotides Oligo-pTa535-1, Oligo-pSc119.2-1, and (GAA)8 as probes, and EST-based molecular markers. The results showed that the partial amphiploid strain consisted of 20 pairs of wheat chromosomes and 7 pairs of Ae. mutica chromosomes, with both wheat 7B chromosomes missing. EST-based molecular marker data suggested that the wheat-Ae. mutica addition line carries the 7T chromosome. Resistance tests indicated that both the partial amphiploid and the 7T addition line were highly resistant to powdery mildew, whereas the wheat control line Chinese Spring was highly susceptible, indicating the presence of a potentially new powdery mildew resistance gene on the Ae. mutica 7T chromosome. The karyotype, FISH patterns, and molecular markers can now be used to identify Ae. mutica chromatin in a wheat background, and the 7T addition could be used as a new powdery mildew resistance source for wheat breeding.


Assuntos
Análise Citogenética/métodos , Resistência à Doença/genética , Doenças das Plantas/genética , Poliploidia , Triticum/genética , Ascomicetos/fisiologia , Bandeamento Cromossômico , Diploide , Hibridização in Situ Fluorescente , Cariótipo , Cariotipagem , Doenças das Plantas/microbiologia , Especificidade da Espécie , Triticum/classificação , Triticum/microbiologia
6.
J Biotechnol ; 189: 48-57, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25240441

RESUMO

With the frequent occurrence of climatic anomalies, spring frost has become a significant limiting factor on wheat production, especially during the reproductive growth stage. A high-throughput sequencing technology was applied and a total of 54 million clean reads that corresponded to 7.44 Gb of total nucleotides were generated. These reads were then de novo assembled into 120,715 unigenes with an average length of 627 bp. Functional annotations were then obtained by aligning all unigenes with public protein databases. In total, 9657 potential EST-SSRs were identified, and 6310 primer pairs for 1329 SSRs were obtained. Meanwhile, a comparison of four tag-based digital gene expression libraries, which was built from the control and cold-treated young spikes were performed. Overall, 526 up-regulated and 489 down-regulated genes were identified, and GO and KEGG pathway analyses of those genes were further conducted. Based on these results, a series of candidate genes involved in cold response pathways were identified, and 12 of them were confirmed by qRT-PCR. The combination of RNA-Seq and digital gene expression analysis in this study provides a powerful approach for investigating the transcriptional changes and obtained a large number of unigenes annotated to public databases.


Assuntos
Transcriptoma/genética , Triticum/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética
7.
Bioorg Chem ; 47: 1-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23474214

RESUMO

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.


Assuntos
Acil-CoA Desidrogenase/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Isovaleril-CoA Desidrogenase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/química , Aminoácidos de Cadeia Ramificada/genética , Animais , Ácidos Graxos/metabolismo , Isovaleril-CoA Desidrogenase/química , Isovaleril-CoA Desidrogenase/genética , Cinética , Fígado/enzimologia , Dados de Sequência Molecular , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Especificidade por Substrato
8.
Biochim Biophys Acta ; 1830(6): 3635-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23428571

RESUMO

BACKGROUND: Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation. METHODS: The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC50 values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy. RESULTS AND CONCLUSIONS: We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC50 values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells. GENERAL SIGNIFICANCE: Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.


Assuntos
Proliferação de Células/efeitos dos fármacos , Difosfonatos , Inibidores Enzimáticos , Corantes Fluorescentes , Ácido Mevalônico/metabolismo , Simulação de Acoplamento Molecular , Animais , Sobrevivência Celular/efeitos dos fármacos , Difosfonatos/síntese química , Difosfonatos/química , Difosfonatos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/enzimologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Ratos
9.
Artigo em Chinês | MEDLINE | ID: mdl-22919749

RESUMO

OBJECTIVE: Comparative and statistical analysis the HPV infection rate between fresh tissue and Paraffin-embedded Specimens of esophageal squamous cell carcinoma,and comparative the testing results with others regions. METHODS: Extracted the total DNA from the novel fresh tissue and Paraffin-embedded Specimens; Detected the DNA by PCR with universal primer and Detected the HPV type with human papilloma virus nucleic acid amplification-based typing detection reagent kit (Hybribio); Compared the statistical result from the different specimens; analyzed the result between different region. RESULTS: HPV infection rate of fresh tissue is 82.6% with HPV16 (34.8%) and HPV18 (34.8%), and paraffin-embedded specimens is 78.2% with HPV16 (30.4%) and HPV18 (17.4%). CONCLUSION: The results provides the first evidence that there wasn't noticeable difference between HPV infection rate of the two specimens. So broader specimen source could be used for HPV testing.


Assuntos
Carcinoma de Células Escamosas/virologia , Neoplasias Esofágicas/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Proteínas do Capsídeo/análise , DNA Viral/isolamento & purificação , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Humanos , Proteínas Oncogênicas Virais/análise , Reação em Cadeia da Polimerase
10.
Chem Commun (Camb) ; 46(29): 5340-2, 2010 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-20544107

RESUMO

The mevalonate pathway is an important drug target for the treatment of cancer and cardiovascular disease. We synthesized and studied a new type of nitrogen-containing bisphosphonate analogs and developed a sensitive end point assay method for enzyme FPPS, which was used for inhibitor screening. One potent FPPS inhibitor was discovered, and the structure-activity relationship of bisphosphonates for the enzyme inactivation was studied.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Ácido Mevalônico/metabolismo , Biocatálise , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-Atividade
11.
J Biotechnol ; 141(3-4): 142-6, 2009 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-19433218

RESUMO

Galactokinase is an ATP-dependent enzyme that catalyzes the phosphorylation of galactose to form galactose-1-phosphate. The defect in human galactokinase can result in the disease of galactosemia. On the other hand, the control of galactose-1-phosphate production by inhibiting galactokinase is a potential therapy for another disease referred to as classic galactosemia. Many pharmaceutically important compounds derive from carbohydrate-containing natural products, and glycorandomization is one of the most efficient approaches for complex secondary metabolites. Therefore, it is important to further understand the interaction between galactokinase and its substrate or substrate analogs. In the present study, we cloned and purified both N- and C-terminal His-tagged rat galactokinase. We then constructed and purified a variety of variant enzymes, which were studied using kinetics with galactose and its analogs as substrates. We found that the binding of the ATP may induce conformational change to the enzyme so that the enzyme can bind galactose specifically. Asp186 was found to be a possible catalytic residue. The mutants were incubated with fluorescent trinitrophenyl-ATP for the characterization of their ATP binding sites. Various other substrate analogs, aminoglycosides and flavanoids were also tested and found to be competitive inhibitors of rat galactokinase.


Assuntos
Ligação Competitiva , Galactoquinase/metabolismo , Galactose/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoglicosídeos/metabolismo , Animais , Flavonoides/metabolismo , Galactoquinase/antagonistas & inibidores , Galactoquinase/genética , Galactose/análogos & derivados , Cinética , Microscopia de Fluorescência , Mutação , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Bioorg Chem ; 37(1): 33-40, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19095280

RESUMO

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Porphobilinogen synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. In the present study, a variety of new substrate analogs and reaction intermediate analogs were synthesized, which were used as probes for studying the active site of rat porphobilinogen synthase. The compounds 1, 3, 6, 9, 14, 16, and 28 were found to be competitive inhibitors of rat porphobilinogen synthase with inhibition constants ranging from 0.96 to 73.04mM. Compounds 7, 10, 12, 13, 15, 17, 18, and 26 were found to be irreversible enzyme inhibitors. For irreversible inhibitors, loose-binding inhibitors were found to give stronger inactivation. The amino group and carboxyl group of the analogs were found to be important for their binding to the enzyme. This study increased our understanding of the active site of porphobilinogen synthase.


Assuntos
Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Sintase do Porfobilinogênio/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Clonagem Molecular , Ativação Enzimática , Inibidores Enzimáticos/síntese química , Cinética , Modelos Químicos , Dados de Sequência Molecular , Sintase do Porfobilinogênio/química , Ratos , Especificidade por Substrato
13.
Bioorg Chem ; 36(5): 241-51, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18760440

RESUMO

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Hydroxymethylbilane synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. We carried out extensive studies of rat hydroxymethylbilane synthase in the present investigation. The enzymatic reaction rate of the holoenzyme was found to be lower than those of the enzyme-intermediate complexes, which corrected the previous theoretical analysis result. Several mutants were constructed, purified and characterized. D44 was found to play an important role in the disassembly of the enzyme-intermediate complexes. E63 and H78 were important for maintaining the activity of the enzyme at high temperature. Four substrate analogs with variation of porphobilinogen side-chain were synthesized and incubated with the enzyme. Three analogs were found to be weak substrates of the enzyme. All four analogs can be used for the preparation of uroporphyrin I analogs.


Assuntos
Histidina/química , Hidroximetilbilano Sintase/metabolismo , Fígado/enzimologia , Porfobilinogênio/química , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Clonagem Molecular , Estabilidade Enzimática , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Hidroximetilbilano Sintase/genética , Hidroximetilbilano Sintase/isolamento & purificação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , Uroporfirinogênios/metabolismo
14.
Biochim Biophys Acta ; 1784(11): 1742-9, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18640292

RESUMO

Mitochondrial trifunctional protein (MTP) catalyzes three consecutive step reactions in the beta-oxidation of long-chain fatty acids, and plays important roles in control and regulation of the beta-oxidation. We overexpressed in E. coli, and purified the MTP as a Mistic fusion protein, which was found to be an alpha(2)beta(2) protein complex and characterized with kinetic studies. Trimetazidine, used for treating chronic stable angina, has been proposed to be an inhibitor of the beta-subunit. We found that a catalytic cysteine residue C105 was labeled by trimetazidine through MS/MS analysis of a trimetazidine-labeled peptide fragment obtained from pepsin digested beta-subunit inactivated by trimetazidine. The MTP beta-subunit was then comparatively studied with monofunctional 3-ketoacyl-CoA thiolase through sequence alignment, site-directed mutagenesis, characterization of variant enzymes with kinetic studies, and homology modeling. The results indicate that the catalytic residues of the MTP beta-subunit are positioned in the active site similarly to those of monofunctional 3-ketoacyl-CoA thiolase.


Assuntos
Descoberta de Drogas , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Clonagem Molecular , Sistemas de Liberação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Cinética , Proteína Mitocondrial Trifuncional , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Transfecção , Trimetazidina/farmacologia
15.
Anal Biochem ; 376(1): 144-50, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18295586

RESUMO

Aminoglycosides are among the most commonly used antibiotics. The intensive use of aminoglycoside antibiotics has led to the problem of food contamination and the development of antibiotic-resistant bacteria. In the present study, we developed an effective method for easy sensitive detection of broad-spectrum aminoglycoside antibiotics. Aminoglycoside 6'-N-acetyltransferase family catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to the 6' amino group of the aminoglycoside, which is one of the most widespread determinants of aminoglycoside resistance. Because acetyl-CoA is naturally present only in living organisms, it is expected that the enzyme can bind with aminoglycoside antibiotics without catalysis in vitro. The enzyme was mutated for the introduction of a cysteine residue to flexible loops close to the binding site, which was then labeled with thio-labeling reagent fluorescein-5-maleimide. The labeled enzymes were characterized with kinetic and binding studies of various known aminoglycoside antibiotics. The binding of the labeled enzyme with aminoglycoside antibiotics causes a conformational change of the enzyme, which subsequently changes the hydrophobicity and hydrophilicity environment of fluorescent labeling reagent resulting in emission of fluorescence. This study provides a sensitive detection method for residual aminoglycoside antibiotics and strategies to screen and discover new effective aminoglycoside antibiotics.


Assuntos
Acetiltransferases/metabolismo , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Fluorescência , Acetiltransferases/química , Acetiltransferases/genética , Sequência de Aminoácidos , Aminoglicosídeos/análise , Aminoglicosídeos/química , Antibacterianos/análise , Antibacterianos/química , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Reprodutibilidade dos Testes , Espectrometria de Fluorescência/métodos
16.
Biochim Biophys Acta ; 1774(12): 1571-81, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17964869

RESUMO

Mevalonate kinase is one of ATP-dependent enzymes in the mevalonate pathway and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. In animal cells, it plays a key role in regulating biosynthesis of cholesterol, while in microorganisms and plants, it is involved in the biosynthesis of isoprenoid derivatives that are one of the largest groups of natural products. Crystal structure and sequence alignment show that a unique disulfide bond exists in mevalonate kinase of thermostable species Methanococcus jannaschii, but not in rat mevalonate kinase. In the present study, we investigated the effect of the disulfide bond in M. jannaschii mevalonate kinase and an engineered disulfide bond in rat mevalonate kinase mutant A141C on the properties of enzymes through characterization of their wild-type and variant enzymes. Our result suggests that the Cys107-Cys281 disulfide bond is important for maintaining the conformation and the thermal activity of M. jannaschii mevalonate kinase. Other interactions could also have contributions. The thiol-titration and fluorescence experiment further indicate that rat mevalonate kinase A141C variant enzyme has a new disulfide bond, which makes the variant protein enhance its thermal activity and resist to urea denaturation.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Dissulfetos/química , Expressão Gênica , Mathanococcus/enzimologia , Mathanococcus/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Desnaturação Proteica , Estrutura Terciária de Proteína , Ratos , Análise de Sequência , Homologia de Sequência de Aminoácidos , Compostos de Sulfidrila/análise , Temperatura
17.
Protein J ; 26(8): 569-76, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17763925

RESUMO

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Urogen III synthase catalyzes a key step in the formation of urogen III, a common intermediate for tetrapyrrolic natural products. In the present study, we cloned, purified, and characterized His-tagged rat urogen III synthase. The mechanism of enzymatic reaction was studied through site-directed mutagenesis of eight highly conserved residues with functional side chains around the active site followed with activity tests. Lys10, Asp17, Glu68, Tyr97, Asn121, Lys147, and His173 have not been studied previously, which were found to be unessential for enzymatic reaction. Tyr168 was identified as an important residue for enzymatic reaction catalyzed by rat urogen III synthase. Molecular modeling suggests the hydroxyl group of Tyr168 side chain is 3.5 A away from the D ring, and is within hydrogen bond distance (1.9 A) with acetate side chain of the D ring.


Assuntos
Histidina/química , Fígado/enzimologia , Uroporfirinogênio III Sintetase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Clonagem Molecular , Cristalografia por Raios X , Biblioteca Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uroporfirinogênio III Sintetase/química , Uroporfirinogênio III Sintetase/genética
18.
Org Lett ; 9(20): 3877-80, 2007 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-17764185

RESUMO

Selective inactivation of cytosolic thiolase by 2-alkynoyl-CoA via its intrinsic isomerase activity was studied, which provides an example for rationally developing mechanism-based inhibitors based on a side activity of the enzyme, and may become a supplemental method for better treatment of cardiovascular disease and cancer.


Assuntos
Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Compostos de Sulfidrila/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Catálise , Linhagem Celular , Ativação Enzimática , Isomerismo , Cinética , Fígado/enzimologia , Estrutura Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Especificidade por Substrato , Espectrometria de Massas em Tandem
19.
Bioorg Med Chem Lett ; 17(11): 3187-90, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17383181

RESUMO

Fluorinated substrate analogs were synthesized and incubated with rat liver 3-hydroxyacyl-CoA dehydrogenase, which reveals that the formation of an enolate intermediate is required for the reaction catalyzed by the enzyme.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acil Coenzima A/química , Coenzima A/química , Animais , Catálise , Coenzima A/síntese química , Ratos , Especificidade por Substrato
20.
Biochim Biophys Acta ; 1760(12): 1874-83, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16952422

RESUMO

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Delta3-Delta2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k(cat) value of 1.0 s(-1) for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Lisina/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Isomerases de Ligação Dupla Carbono-Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Primers do DNA , Dodecenoil-CoA Isomerase , Lisina/genética , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos
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