Comparative study of the production of coumarins and furanocoumarins in three Ruteae species

Within specialized metabolites, coumarins and furanocoumarins represent a wide group of structurally diverse compounds and are specially produced in plants belonging to the Rutaceae family. Here we performed the furanocoumarin and coumarin-targeted chemical characterization of three Ruteae species collected from Algeria. Detection and quantification of 27 coumarins and furanocoumarins extracted from stems and leaves was carried out by UHPLC-MS. We highlighted significant chemical differences between these plants. Ruta chalepensis L. is the highest producer with 24.83 mmol/g dry material in stems and 15.70 mmol/g dry materials in leaves while Haplophyllum tuberculatum (Forsk.) is the lowest producer. We also showed a surprising chemical diversity between R. chalepensis L and R. angustifolia Pers. This chemical diversity might, therefore, be a helpful tool for phylogenetic identification of plants

Comparative study of the production of coumarins and furanocoumarins in three Ruteae species

137-142Within specialized metabolites, coumarins and furanocoumarins represent a wide group of structurally diverse compounds and are specially produced in plants belonging to the Rutaceae family. Here we performed the furanocoumarin and coumarin-targeted chemical characterization of three Ruteae species collected from Algeria. Detection and quantification of 27 coumarins and furanocoumarins extracted from stems and leaves was carried out by UHPLC-MS. We highlighted significant chemical differences between these plants. Ruta chalepensis L. is the highest producer with 24.83 mmol/g dry material in stems and 15.70 mmol/g dry materials in leaves while Haplophyllum tuberculatum (Forsk.) is the lowest producer. We also showed a surprising chemical diversity between R. chalepensis L and R. angustifolia Pers. This chemical diversity might, therefore, be a helpful tool for phylogenetic identification of plants

An extensive (co-)expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Arabidopsis thaliana</it>, 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.</p> <p>Conclusion</p> <p>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.</p

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

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.[br/] 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.[br/] 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

The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants

International audienceThe 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

Mécanisme et spécificité structurale des Méthionine sulfoxyde réductases (Msr) de Neisseria meningitidis et rôle du métal dans les MsrB

Methionine sulfoxide reductases (Msr) allow cells to restore function of oxidized proteins on their methionine residues. In the first part, the catalytic mechanism of the MsrA and MsrB of the PilB protein from the bacterial pathogen Neisseria meningitidis has been studied. The two structurally unrelated classes of Msrs display a similar three step catalytic mechanism including the formation of a sulfenic acid intermediate followed by the formation of an intra disulfide bond which is reduced by thioredoxin (Trx). The two classes of Msrs conversely present an opposite stereoselectivity towards the sulfoxide function. In the second part, the three steps of the MsrBs catalytic mechanism have been kinetically characterized. The study of the role of the catalytic amino acid, the characterisation of the MsrB-Trx interaction and, the study of the role of the coordinated metal have also been investigated.Les méthionine sulfoxyde réductases (Msr) permettent de restaurer la fonction des protéines oxydées sur leur résidus Methionine. Dans un premier temps, le mécanisme catalytique de la MsrA et de la MsrB de la protéine PilB de la bactérie pathogène Neisseria meningitidis a été étudié. Les deux classes de Msr, qui sont structuralement différentes, partagent un même mécanisme catalytique en trois étapes avec formation d'un intermédiaire acide sulfénique suivie de la formation d'un pont disulfure intra moléculaire qui est réduit par la thiorédoxine (Trx). Elles présentent en revanche une stéréospécificité de substrat inverse vis-à-vis de la fonction sulfoxyde. Dans un deuxième temps, les trois étapes du mécanisme catalytique de la MsrB ont été caractérisées au niveau cinétique. L'étude du rôle des acides aminés du site actif dans la catalyse, la caractérisation biochimique de l'interaction MsrB-Trx et, enfin, l'étude du rôle du métal coordiné ont également été abordées

Mécanisme et spécificité structurale des Méthionine sulfoxyde réductases (Msr) de

Methionine sulfoxide reductases (Msr) allow cells to restore function of oxidized proteins on their methionine residues. In the first part, the catalytic mechanism of the MsrA and MsrB of the PilB protein from the bacterial pathogen Neisseria meningitidis has been studied. The two structurally unrelated classes of Msrs display a similar three step catalytic mechanism including the formation of a sulfenic acid intermediate followed by the formation of an intra disulfide bond which is reduced by thioredoxin (Trx). The two classes of Msrs conversely present an opposite stereoselectivity towards the sulfoxide function. In the second part, the three steps of the MsrBs catalytic mechanism have been kinetically characterized. The study of the role of the catalytic amino acid, the characterisation of the MsrB-Trx interaction and, the study of the role of the coordinated metal have also been investigated.Les méthionine sulfoxyde réductases (Msr) permettent de restaurer la fonction des protéines oxydées sur leur résidus Methionine. Dans un premier temps, le mécanisme catalytique de la MsrA et de la MsrB de la protéine PilB de la bactérie pathogène Neisseria meningitidis a été étudié. Les deux classes de Msr, qui sont structuralement différentes, partagent un même mécanisme catalytique en trois étapes avec formation d'un intermédiaire acide sulfénique suivie de la formation d'un pont disulfure intra moléculaire qui est réduit par la thiorédoxine (Trx). Elles présentent en revanche une stéréospécificité de substrat inverse vis-à-vis de la fonction sulfoxyde. Dans un deuxième temps, les trois étapes du mécanisme catalytique de la MsrB ont été caractérisées au niveau cinétique. L'étude du rôle des acides aminés du site actif dans la catalyse, la caractérisation biochimique de l'interaction MsrB-Trx et, enfin, l'étude du rôle du métal coordiné ont également été abordées

Mécanisme et spécificité structurale des Méthionine sulfoxyde réductases (Msr) de Neisseria meningitidis et rôle du métal dans les MsrB

Les méthionine sulfoxyde réductases (Msr) permettent de restaurer la fonction des protéines oxydées sur leur résidus Methionine. Dans un premier temps, le mécanisme catalytique de la MsrA et de la MsrB de la protéine PilB de la bactérie pathogène Neisseria meningitidis a été étudié. Les deux classes de Msr, qui sont structuralement différentes, partagent un même mécanisme catalytique en trois étapes avec formation d'un intermédiaire acide sulfénique suivie de la formation d'un pont disulfure intra moléculaire qui est réduit par la thiorédoxine (Trx). Elles présentent en revanche une stéréospécificité de substrat inverse vis-à-vis de la fonction sulfoxyde. Dans un deuxième temps, les trois étapes du mécanisme catalytique de la MsrB ont été caractérisées au niveau cinétique. L'étude du rôle des acides aminés du site actif dans la catalyse, la caractérisation biochimique de l'interaction MsrB-Trx et, enfin, l'étude du rôle du métal coordiné ont également été abordées.Methionine sulfoxide reductases (Msr) allow cells to restore function of oxidized proteins on their methionine residues. In the first part, the catalytic mechanism of the MsrA and MsrB of the PilB protein from the bacterial pathogen Neisseria meningitidis has been studied. The two structurally unrelated classes of Msrs display a similar three step catalytic mechanism including the formation of a sulfenic acid intermediate followed by the formation of an intra disulfide bond which is reduced by thioredoxin (Trx). The two classes of Msrs conversely present an opposite stereoselectivity towards the sulfoxide function. In the second part, the three steps of the MsrBs catalytic mechanism have been kinetically characterized. The study of the role of the catalytic amino acid, the characterisation of the MsrB-Trx interaction and, the study of the role of the coordinated metal have also been investigated.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Characterization of the amino acids from Neisseria meningitidis methionine sulfoxide reductase B involved in the chemical catalysis and substrate specificity of the reductase step.

Methionine sulfoxide reductases (Msrs) are antioxidant repair enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The Msr family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the S and R isomers of the sulfoxide function, respectively. Both classes of Msr share a similar three-step chemical mechanism involving first a reductase step that leads to the formation of a sulfenic acid intermediate. In this study, the invariant amino acids of Neisseria meningitidis MsrB involved in the reductase step catalysis and in substrate binding have been characterized by the structure-function relationship approach. Altogether the results show the following: 1) formation of the MsrB-substrate complex leads to an activation of the catalytic Cys-117 characterized by a decreased pKapp of approximately 2.7 pH units; 2) the catalytic active MsrB form is the Cys-117-/His-103+ species with a pKapp of 6.6 and 8.3, respectively; 3) His-103 and to a lesser extent His-100, Asn-119, and Thr-26 (via a water molecule) participate in the stabilization of the polarized form of the sulfoxide function and of the transition state; and 4) Trp-65 is essential for the catalytic efficiency of the reductase step by optimizing the position of the substrate in the active site. A scenario for the reductase step is proposed and discussed in comparison with that of MsrA

The distribution of coumarins and furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways

In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes.In this study, coumarin and furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and furanocoumarin content varieties.Citrus varieties identified in this study with low coumarin and furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules