RESUMO
Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple (13)C-, (15)N-, (17)O-, and (18)O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.
Assuntos
Bacillus subtilis/química , Escherichia coli/química , Escherichia coli/enzimologia , Flavoproteínas/química , Marcação por Isótopo/métodos , Pteridinas/química , Pteridinas/síntese química , Purinas/química , Riboflavina Sintase/química , Riboflavina/química , Biotransformação , Ligantes , Riboflavina Sintase/metabolismoRESUMO
The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.
Assuntos
Isótopos de Carbono/química , Transferases Intramoleculares/química , Olea/química , Triterpenos Pentacíclicos/biossíntese , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/síntese química , Esqualeno/análogos & derivados , Esqualeno/química , Estearatos/síntese química , Taraxacum/química , Triterpenos/síntese química , Sequência de Aminoácidos , Ciclização , Espectroscopia de Ressonância Magnética , Esqualeno/síntese química , Estearatos/química , Triterpenos/químicaRESUMO
Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K(i) of 70microM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K(i) 38microM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve pi-pi stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.
Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Antituberculosos/química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Indóis/química , Estrutura Molecular , Mycobacterium tuberculosis/enzimologiaRESUMO
Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of 13CO2 for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by 13C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.
Assuntos
Antimaláricos/metabolismo , Artemisia annua/metabolismo , Artemisininas/metabolismo , Vias Biossintéticas , Ácido Mevalônico/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Antimaláricos/química , Antimaláricos/isolamento & purificação , Artemisia annua/química , Artemisininas/química , Artemisininas/isolamento & purificação , Dióxido de Carbono/metabolismo , Isótopos de Carbono , Malária/tratamento farmacológico , Estrutura Molecular , Fitoterapia , Sesquiterpenos Policíclicos , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/químicaRESUMO
We screened thirty-four methanolic plant extracts for inhibition of the constitutive nuclear factor kappaB (NFkappaB) activity by a NFkappaB-luciferase reporter gene assay. Strong inhibition of NFkappaB activity was found in extracts of leaf and rhizome from Nuphar lutea L. SM. (Nuphar). The inhibitory action was narrowed down to a mixture of thionupharidines and/or thionuphlutidines that were identified in chromatography fractions by one- and two-dimensional NMR analysis. Dimeric sesquiterpene thioalkaloids were identified as the major components of the mixture. The Nuphar alkaloids mixture (NUP) showed a dose dependent inhibition of NFkappaB activity in a luciferase reporter gene assay as well as reduction of nuclear NFkappaB subunits expression as tested by western blots and immunohistochemistry. Decreased DNA binding was demonstrated in electro mobility shift assays. NUP inhibited both inducible and constitutive NFkappaB activation and affected the canonical and alternative pathways. Suppression of NFkappaB was not cell type specific. Induction of apoptosis by the alkaloid mixture was demonstrated by time-dependent and dose-dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of the active mixture with cisplatin and etoposide was demonstrated. Overall, our results show that NUP inhibits the NFkappaB pathway and acts as a sensitizer to conventional chemotherapy, enabling the search for its specific target and application against cancer and inflammation.
Assuntos
Alcaloides/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Etoposídeo/farmacologia , NF-kappa B/antagonistas & inibidores , Nuphar/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Alcaloides/administração & dosagem , Alcaloides/isolamento & purificação , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Etoposídeo/administração & dosagem , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Espectroscopia de Ressonância Magnética , Metanol/química , NF-kappa B/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Compostos de Sulfidrila/isolamento & purificação , Compostos de Sulfidrila/farmacologiaRESUMO
Enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are therapeutic targets for the treatment of important infectious diseases. Whereas this pathway is absent in humans, it is used by plants, many eubacteria and apicomplexan protozoa, including major human pathogens such as Plasmodium falciparum and Mycobacterium tuberculosis. Herein, we report on the design, preparation and biological evaluation of a new series of ligands for IspE protein, a kinase from this pathway. These inhibitors were developed for the inhibition of IspE from Escherichia coli, using structure-based design approaches. Structure-activity relationships (SARs) and a co-crystal structure of Aquifex aeolicus IspE bound to a representative inhibitor validate the proposed binding mode. The crystal structure shows that the ligand binds in the substrate-rather than the adenosine 5'-triphosphate (ATP)-binding pocket. As predicted, a cyclopropyl substituent occupies a small cavity not used by the substrate. The optimal volume occupancy of this cavity is explored in detail. In the co-crystal structure, a diphosphate anion binds to the Gly-rich loop, which normally accepts the triphosphate moiety of ATP. This structure provides useful insights for future structure-based developments of inhibitors for the parasite enzymes.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Modelos Biológicos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/química , Concentração Inibidora 50 , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Relação Estrutura-AtividadeRESUMO
Rapid progress in instrumentation and software made nuclear magnetic resonance spectroscopy (NMR) one of the most powerful analytical methods in biological sciences. Whereas the development of multidimensional NMR pulse sequences is an ongoing process, a small subset of two-dimensional NMR experiments is typically sufficient for the rapid structure determination of small metabolites. The use of sophisticated three- and four-dimensional NMR experiments enables the determination of the three-dimensional structures of proteins with a molecular weight up to 100 kDa, and solution structures of more than 100 plant proteins have been established by NMR spectroscopy. NMR has also been introduced to the emerging field of metabolomics where it can provide unbiased information about metabolite profiles of plant extracts. In recent times, high-resolution NMR has become a key technology for the elucidation of biosynthetic pathways and metabolite flux via quantitative assessment of multiple isotopologues. This review summarizes some of the recent advances of high-resolution NMR spectroscopy in the field of plant sciences.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Plantas/química , Plantas/metabolismo , Fenômenos Bioquímicos , Bioquímica , Produtos Biológicos/biossíntese , Produtos Biológicos/química , Estrutura MolecularRESUMO
3,4-Dihydroxy-2-butanone 4-phosphate synthase, 6,7-dimethyl-8-ribityllumazine synthase, and riboflavin synthase of the riboflavin biosynthetic pathway are potential targets for novel antiinfective drugs. This article describes a platform for high-throughput screening for inhibitors of these enzymes. The assays can be monitored photometrically and have been shown to be robust, as indicated by Z factors 0.87. A (13)C NMR assay for hit verification of 3,4-dihydroxy-2-butanone 4-phosphate synthase inhibitors is also reported.
Assuntos
Bacillus subtilis/enzimologia , Técnicas Analíticas Microfluídicas/métodos , Riboflavina Sintase/metabolismo , Riboflavina/antagonistas & inibidores , Riboflavina/biossíntese , Anti-Infecciosos/metabolismo , Bacillus subtilis/genética , Vias Biossintéticas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Estrutura Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Pteridinas , Riboflavina/química , Riboflavina Sintase/químicaAssuntos
Antimaláricos/síntese química , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/síntese química , Proteínas de Escherichia coli/antagonistas & inibidores , Ácido Mevalônico/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Terpenos/química , Antimaláricos/uso terapêutico , Ligação Competitiva , Desenho de Fármacos , Inibidores Enzimáticos/uso terapêutico , Proteínas de Escherichia coli/química , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/química , Ácido Mevalônico/metabolismo , Fosfatos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Terpenos/metabolismo , Thermus thermophilus/enzimologia , Transferases/antagonistas & inibidores , Transferases/químicaRESUMO
The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Fósforo-Oxigênio Liases/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Aldose-Cetose Isomerases/biossíntese , Anti-Infecciosos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/biossíntese , Testes de Sensibilidade Microbiana , Estrutura Molecular , Complexos Multienzimáticos/biossíntese , Oxirredutases/biossíntese , Fósforo-Oxigênio Liases/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de TempoAssuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Corantes Fluorescentes/farmacologia , Fósforo-Oxigênio Liases/antagonistas & inibidores , Terpenos/metabolismo , Antimaláricos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Corantes Fluorescentes/síntese química , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fósforo-Oxigênio Liases/químicaRESUMO
A high-throughput screening method based on the competitive binding of a lumazine synthase inhibitor and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase was developed. This assay is sensitive, simple, and robust. During assay development, all of the known active inhibitors tested were positively identified. Preliminary high-throughput screening in 384-well format resulted in a Z factor of 0.7. The approach utilizes a thermodynamic assay to bypass the problems associated with the instabilities of both lumazine synthase substrates that complicate the use of a kinetic assay in a high-throughput format, and it removes the time element from the assay, thus simplifying the procedure.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Complexos Multienzimáticos/antagonistas & inibidores , Riboflavina/química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Ligantes , Schizosaccharomyces/enzimologia , Espectrometria de FluorescênciaRESUMO
The discovery of a distinct metabolic pathway, the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for isoprenoid precursor biosynthesis, in eubacteria and apicomplexan parasites has revealed a new set of potential drug targets. The emphasis of research on this pathway has been on delineating the intermediates and the biochemical and structural characterization of component enzymes. Two new monoclinic crystal forms of recombinant Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECP) synthase cocrystallized with (i) CMP and (ii) CMP and MECP show well defined electron density at the subunit interface suggestive of an isoprenoid-like ligand. 31P NMR analysis of the recombinant protein sample indicates the presence of bound diphosphate species and electrospray mass spectrometry identifies a mixture of isopentenyl diphosphate (and/or dimethylallyl diphosphate), geranyl diphosphate and farnesyl diphosphate in an approximate ratio of 1:4:2. The most prevalent species, geranyl diphosphate, was successfully modelled into the electron density, revealing the important protein-ligand interactions that stabilize binding of the isoprenoid. The observation that MECP synthase binds three metabolites that are produced by enzymes two, three and four stages downstream in isoprenoid biosynthesis suggests that feedback regulation of the non-mevalonate pathway is possible.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Fósforo-Oxigênio Liases/química , Terpenos/química , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados como Assunto , Elétrons , Íons , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Químicos , Modelos Moleculares , Fosfatos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray , ZincoRESUMO
An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium. Growth of the recombinant strain in medium supplemented with [U-13C6]glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N. The yield of [U-13C13]-6,7-dimethyl-8-ribityllumazine based on [U-13C6]glucose was 25 mg/g. Fermentation with [1-13C1]-, [2-13C1]-, or [3-13C1]glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms. The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine. Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach.
Assuntos
Bacillus subtilis/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Pteridinas/metabolismo , Riboflavina/biossíntese , Bacillus subtilis/enzimologia , Isótopos de Carbono , Escherichia coli/genética , Engenharia Genética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Isótopos de Nitrogênio , Pteridinas/isolamento & purificaçãoRESUMO
Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.
Assuntos
Aldeído Liases/metabolismo , Di-Hidropteroato Sintase/metabolismo , Difosfotransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/enzimologia , Tetra-Hidrofolatos/metabolismo , Aldeído Liases/análise , Aldeído Liases/genética , Proteínas de Ligação a DNA/genética , Di-Hidropteroato Sintase/análise , Di-Hidropteroato Sintase/genética , Difosfotransferases/análise , Difosfotransferases/genética , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.
Assuntos
Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/metabolismo , Riboflavina/biossíntese , Sequência de Aminoácidos , Arabidopsis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Fenômenos Bioquímicos , Bioquímica , Proteínas de Transporte/química , Clonagem Molecular , DNA/metabolismo , Enzimas de Restrição do DNA/farmacologia , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Evolução Molecular , GTP Cicloidrolase/química , Teste de Complementação Genética , Guanosina Trifosfato/química , Cinética , Espectroscopia de Ressonância Magnética , Proteínas Ligantes de Maltose , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/química , Fases de Leitura Aberta , Filogenia , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Atômica , Desidrogenase do Álcool de Açúcar/química , Fatores de Tempo , Zinco/químicaRESUMO
Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.
Assuntos
Aldeído Liases/genética , Arabidopsis/enzimologia , Ácido Fólico/biossíntese , Solanum lycopersicum/enzimologia , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Ácido Fólico/química , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Racemases e Epimerases/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Root cultures of Swertia chirata (Gentianaceae) were grown with supplements of [1-13C]glucose, [U-13C6]glucose or [carboxy-13C]shikimic acid. 1,3,5,8-Tetrahydroxyxanthone was isolated and analysed by quantitative NMR analysis. The observed isotopomer distribution shows that 1,3,5,8-tetrahydroxyxanthone is biosynthesized via a polyketide-type pathway. The starter unit, 3-hydroxybenzoyl-CoA, is obtained from an early shikimate pathway intermediate. Phenylalanine, cinnamic acid and benzoic acid were ruled out as intermediates.
Assuntos
Gentianaceae/metabolismo , Fenilalanina/metabolismo , Xantenos/metabolismo , Xantonas , Acil Coenzima A/metabolismo , Ácido Benzoico/química , Ácido Benzoico/metabolismo , Isótopos de Carbono , Cinamatos/química , Cinamatos/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfoenolpiruvato/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Ácido Chiquímico/análogos & derivados , Ácido Chiquímico/metabolismo , Fosfatos Açúcares/metabolismo , Xantenos/química , Xantenos/isolamento & purificaçãoRESUMO
Cut sprouts of Hypericum perforatum were proffered solutions containing [1-(13)C]glucose or [U-(13)C(6)]glucose. Hyperforin was isolated and analyzed by quantitative NMR spectroscopy. The labeling patterns show that the biosynthesis of hyperforin involves five isoprenoid moieties, which are derived entirely or predominantly (>98%) via the deoxyxylulose phosphate pathway. The phloroglucinol moiety is generated via a polyketide type mechanism.
Assuntos
Hypericum/metabolismo , Terpenos/metabolismo , Compostos Bicíclicos com Pontes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Floroglucinol/análogos & derivadosRESUMO
GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of approximately 26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying approximately 50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.