RESUMO
Plants produce â¼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.
Assuntos
Angelica/genética , Citrus paradisi/genética , Evolução Molecular , Furocumarinas/biossíntese , Proteínas de Plantas/genética , Prenilação , Angelica/metabolismo , Citrus paradisi/metabolismo , Filogenia , Proteínas de Plantas/metabolismoRESUMO
Coumarins are phytochemicals occurring in the plant kingdom, which biosynthesis is induced under various stress factors. They belong to the wide class of specialized metabolites well known for their beneficial properties. Due to their high and wide biological activities, coumarins are important not only for the survival of plants in changing environmental conditions, but are of great importance in the pharmaceutical industry and are an active source for drug development. The identification of coumarins from natural sources has been reported for different plant species including a model plant Arabidopsis thaliana. In our previous work, we demonstrated a presence of naturally occurring intraspecies variation in the concentrations of scopoletin and its glycoside, scopolin, the major coumarins accumulating in Arabidopsis roots. Here, we expanded this work by examining a larger group of 28 Arabidopsis natural populations (called accessions) and by extracting and analysing coumarins from two different types of tissues-roots and leaves. In the current work, by quantifying the coumarin content in plant extracts with ultra-high-performance liquid chromatography coupled with a mass spectrometry analysis (UHPLC-MS), we detected a significant natural variation in the content of simple coumarins like scopoletin, umbelliferone and esculetin together with their glycosides: scopolin, skimmin and esculin, respectively. Increasing our knowledge of coumarin accumulation in Arabidopsis natural populations, might be beneficial for the future discovery of physiological mechanisms of action of various alleles involved in their biosynthesis. A better understanding of biosynthetic pathways of biologically active compounds is the prerequisite step in undertaking a metabolic engineering research.
Assuntos
Arabidopsis/metabolismo , Cumarínicos/análise , Espectrometria de Massas , Raízes de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Cumarínicos/metabolismoRESUMO
Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co-localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3-CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α-ketoglutarate-dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p-coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Pastinaca/genética , Pastinaca/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Furocumarinas/metabolismo , Hibridização in Situ Fluorescente , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Iron deficiency is a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under iron deficiency. An essential enzyme for the biosynthesis of the major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6'-Hydroxylase1 (F6'H1), which belongs to a large enzyme family of the 2-oxoglutarate and Fe2+-dependent dioxygenases. We have functionally characterized another enzyme of this family, which is a close homologue of F6'H1 and is encoded by a strongly iron-responsive gene, At3g12900. We purified At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin, via hydroxylation at the C8 position, and that it thus functions as a scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves, followed by metabolite profiling and biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various iron regimes. Our results indicate that S8H is involved in coumarin biosynthesis, as part of mechanisms used by plants to assimilate iron.
Assuntos
Arabidopsis/genética , Cumarínicos/metabolismo , Deficiências de Ferro , Arabidopsis/enzimologia , Escopoletina/metabolismo , Metabolismo SecundárioRESUMO
In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.
Assuntos
Dimetilaliltranstransferase/metabolismo , Evolução Molecular , Furocumarinas/biossíntese , Pastinaca/metabolismo , Proteínas de Plantas/metabolismo , Catharanthus/genética , Membrana Celular/metabolismo , Clonagem Molecular , Cumarínicos/metabolismo , Dimetilaliltranstransferase/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Especificidade por Substrato , Nicotiana/genética , Umbeliferonas/biossíntese , Umbeliferonas/metabolismoRESUMO
Furanocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor of all furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4-coumaroyl CoA 2'-hydroxylase gene in Nicotiana benthamiana, which does not produce furanocoumarins, resulted in formation of demethylsuberosin, indicating that furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants.
Assuntos
Dimetilaliltranstransferase/metabolismo , Furocumarinas/metabolismo , Regulação Enzimológica da Expressão Gênica , Petroselinum/enzimologia , Ruta/enzimologia , Sequência de Bases , Cumarínicos/química , Cumarínicos/metabolismo , Dimetilaliltranstransferase/genética , Furocumarinas/química , Regulação da Expressão Gênica de Plantas , Genes Reporter , Dados de Sequência Molecular , Cebolas/citologia , Cebolas/genética , Cebolas/metabolismo , Especificidade de Órgãos , Petroselinum/genética , Petroselinum/efeitos da radiação , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Ruta/genética , Ruta/efeitos da radiação , Análise de Sequência de DNA , Especificidade por Substrato , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/efeitos da radiação , Raios Ultravioleta , Umbeliferonas/química , Umbeliferonas/metabolismoRESUMO
BACKGROUND: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects. RESULTS: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. CONCLUSION: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.
Assuntos
Apiaceae/enzimologia , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Duplicação Gênica , Sequência de Aminoácidos , Apiaceae/química , Apiaceae/classificação , Apiaceae/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.
Assuntos
Citrus/enzimologia , Dimetilaliltranstransferase/metabolismo , Difosfatos/metabolismo , Diterpenos/metabolismo , Citrus/genética , Dimetilaliltranstransferase/genética , Dados de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Ruta , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: Scopoletin and its glucoside scopolin are important secondary metabolites synthesized in plants as a defense mechanism against various environmental stresses. They belong to coumarins, a class of phytochemicals with significant biological activities that is widely used in medical application and cosmetics industry. Although numerous studies showed that a variety of coumarins occurs naturally in several plant species, the details of coumarins biosynthesis and its regulation is not well understood. It was shown previously that coumarins (predominantly scopolin and scopoletin) occur in Arabidopsis thaliana (Arabidopsis) roots, but until now nothing is known about natural variation of their accumulation in this model plant. Therefore, the genetic architecture of coumarins biosynthesis in Arabidopsis has not been studied before. RESULTS: Here, the variation in scopolin and scopoletin content was assessed by comparing seven Arabidopsis accessions. Subsequently, a quantitative trait locus (QTL) mapping was performed with an Advanced Intercross Recombinant Inbred Lines (AI-RILs) mapping population EstC (Est-1 × Col). In order to reveal the genetic basis of both scopolin and scopoletin biosynthesis, two sets of methanol extracts were made from Arabidopsis roots and one set was additionally subjected to enzymatic hydrolysis prior to quantification done by high-performance liquid chromatography (HPLC). We identified one QTL for scopolin and five QTLs for scopoletin accumulation. The identified QTLs explained 13.86% and 37.60% of the observed phenotypic variation in scopolin and scopoletin content, respectively. In silico analysis of genes located in the associated QTL intervals identified a number of possible candidate genes involved in coumarins biosynthesis. CONCLUSIONS: Together, our results demonstrate for the first time that Arabidopsis is an excellent model for studying the genetic and molecular basis of natural variation in coumarins biosynthesis in plants. It additionally provides a basis for fine mapping and cloning of the genes involved in scopolin and scopoletin biosynthesis. Importantly, we have identified new loci for this biosynthetic process.
Assuntos
Arabidopsis/genética , Cumarínicos/metabolismo , Glucosídeos/metabolismo , Locos de Características Quantitativas/genética , Escopoletina/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Mapeamento Cromossômico , Cumarínicos/química , Glucosídeos/química , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Escopoletina/química , Metabolismo SecundárioRESUMO
Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.
Assuntos
Dioxigenases/metabolismo , Ruta/enzimologia , Umbeliferonas/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Cumarínicos/análise , Cumarínicos/isolamento & purificação , Cumarínicos/metabolismo , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Dioxigenases/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Furocumarinas/metabolismo , Furocumarinas/farmacologia , Expressão Gênica/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Caules de Planta/química , Caules de Planta/enzimologia , Caules de Planta/genética , RNA de Plantas/metabolismo , Ruta/química , Ruta/genética , Escopoletina/análise , Escopoletina/metabolismo , Alinhamento de Sequência , Nicotiana/enzimologia , Nicotiana/genética , Transgenes , Umbeliferonas/análise , Umbeliferonas/metabolismoRESUMO
BACKGROUND: Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis. RESULTS: In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn't affect the synthesis of scopoletin. CONCLUSIONS: The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.
Assuntos
Ácido Clorogênico/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/biossíntese , Oxigenases de Função Mista/metabolismo , Ruta/genética , Sequência de Aminoácidos , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Vetores Genéticos , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Folhas de Planta/genética , Ruta/enzimologia , Escopoletina/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Furanocoumarins are defense molecules mainly described in four plant families that are phylogenetically distant. Molecular characterization of the biosynthetic pathway has been started for many years in Apiaceae and Rutaceae. The results obtained thus far in Apiaceae indicated a major role of cytochromes P450 (P450s) in the CYP71 family. In the present work, we describe the importance of another subfamily of P450s, CYP82D, identified by using a deep analysis of the citrus (Rutaceae) genome and microarray database. CYP82D64 is able to hydroxylate xanthotoxin to generate 5-OH-xanthotoxin. Minor and limited amino acid changes in the CYP82D64 coding sequence between Citrus paradisi and Citrus hystrix provide the enzyme in the latter with the ability to hydroxylate herniarin, but with low efficiency. The kinetic constants of the enzyme are consistent with those of other enzymes of this type in plants and indicate that it may be the physiological substrate. The activity of the enzyme is identical to that of CYP71AZ6 identified in parsnip, showing possible evolutionary convergence between these two families of plants. It is highly possible that these molecules are derived from the synthesis of ubiquitous coumarins throughout the plant kingdom.
Assuntos
Citrus/genética , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Furocumarinas/química , Proteínas de Plantas/genética , Sequência de Aminoácidos , Citrus/química , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMO
The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale.
Assuntos
Ipomoea batatas , Lipase/metabolismo , Proteínas de Plantas/metabolismo , Ácido Quínico/análogos & derivados , Proteínas Recombinantes/metabolismo , Ipomoea batatas/enzimologia , Ipomoea batatas/genética , Lipase/química , Lipase/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ácido Quínico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01488-x.
RESUMO
BACKGROUND: Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s) is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. RESULTS: We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. CONCLUSION: The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Família Multigênica/genética , Arabidopsis/efeitos dos fármacos , Ciclopentanos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Vetores Genéticos , Internet , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/biossíntese , Reguladores de Crescimento de Plantas/farmacologia , Plastídeos/enzimologia , Plastídeos/genética , Reprodutibilidade dos Testes , Triterpenos/metabolismoRESUMO
The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
RESUMO
The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.
Assuntos
Oxirredutases/metabolismo , Sequência de Aminoácidos , Cinética , Metais/metabolismo , Metionina Sulfóxido Redutases , Dados de Sequência Molecular , Oxirredutases/química , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Methionine sulfoxide reductases B (MsrBs) catalyze the reduction of methionine-R-sulfoxide via a three-step chemical mechanism including a reductase step, formation of an intradisulfide bond followed by a thioredoxin recycling process. Fifty percent of the MsrBs, including the Escherichia coli enzyme, possess a metal binding site composed of two CXXC motifs of unknown function. It is located on the opposite side of the active site. The overexpressed E. coli MsrB tightly binds one atom of zinc/iron. Substitution of the cysteines of E. coli MsrB results in complete loss of bound metal and reductase activity, and leads to a low-structured conformation of the protein as shown by CD, fluorescence, and DSC experiments. Introduction of the two CXXC motifs in Neisseria meningitidis MsrB domain leads to a MsrB that tightly binds one atom of zinc/iron, shows a strongly increased thermal stability and displays a reductase activity similar to that of the wild-type but lacking thioredoxin recycling activity. These results demonstrate the stabilizing effect of the metal and the existence of a preformed metal binding site in the nonbound metal MsrB. The data also indicate that metal binding to N. meningitidis MsrB induces subtle structural modifications, which prevent formation of a competent binary complex between oxidized MsrB and reduced thioredoxin but not between reduced MsrB and substrate. The fact that the E. coli and the N. meningitidis MsrBs exhibit a similar thermal stability suggests the existence of other structural factors in the nonbound metal MsrBs that compensate the metal bound stabilizing effect.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Metais/química , Neisseria meningitidis/enzimologia , Oxirredutases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Catálise , Cisteína/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferro/química , Metionina Sulfóxido Redutases , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/metabolismo , Alinhamento de Sequência , Zinco/químicaRESUMO
Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the "grapefruit juice effect". Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.