Plant cytochrome P450 monooxygenases were long considered to be highly substrate-specific, regioselective and stereoselective enzymes, in this respect differing from their animal counterparts. The functional data that have recently... more
Plant cytochrome P450 monooxygenases were long considered to be highly substrate-specific, regioselective and stereoselective enzymes, in this respect differing from their animal counterparts. The functional data that have recently accumulated clearly counter this initial dogma. Highly promiscuous P450 enzymes have now been reported, mainly in terpenoid pathways with functions in plant adaptation, but also some very versatile xenobiotic/herbicide metabolizers. An overlap and predictable interference between endogenous and herbicide metabolism are starting to emerge. Both substrate preference and permissiveness vary between plant P450 families, with high promiscuity seemingly favoring retention of gene duplicates and evolutionary blooms. Yet significant promiscuity can also be observed in the families under high negative selection and with essential functions, usually enhanced after gene duplication. The strategies so far implemented, to systematically explore P450 catalytic capacity...
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SummaryIn order to obtain plant markers of chemical stress and possible tools for the bio‐monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic‐inducible P450 oxygenases. O‐dealkylation of... more
SummaryIn order to obtain plant markers of chemical stress and possible tools for the bio‐monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic‐inducible P450 oxygenases. O‐dealkylation of 7‐ethoxycoumarin is catalysed in Helianthus tuberosus by cytochromes P450 strongly inducible by a wide range of xenobiotics. Therefore, a 7‐ethoxycoumarin O‐de‐ethylase (ECOD) was purified from induced tuber tissues (Batard et al. 1995). A primer designed from an internal peptide sequence, but also corresponding to a conserved P450 haem‐binding region, led to the generation of a gene‐specific probe corresponding to a P450 strongly inducible by aminopyrine. Two partial and 98% identical coding sequences were isolated from a cDNA library prepared from aminopyrine‐induced tuber. A full‐length cDNA was reconstituted by 5′‐RACE elongation. The protein deduced from this full‐length sequence, with 41.1% amino acid identity to CYP76A1 and high phylogenetic relationship to other CYP76s, was termed CYP76B1. CYP76B1 was expressed in yeast. Microsomes from the transformed yeast catalysed the NADPH‐dependent O‐deakylation of 7‐ethoxycoumarin. However, protein sequence as well as enzymological data indicated that CYP76B1 does not correspond to the purified ECOD protein. These results confirm previous data and demonstrate that several P450s in H. tuberosus are capable of actively catalysing the O‐de‐ethylation of ethoxycoumarin. Determination of the steady‐state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments. CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.
Research Interests: Biochemistry, Plant Biology, Biology, Medicine, Plant, and 4 moreYeast, Molecular cloning, Xenobiotic, and Helianthus
Metabolism is a key component of herbicide efficiency, selectivity, and in the appearance of resistance. In many cases, oxygen transfer is the first, obligate step in the biotransformation process. These reactions are both slow and... more
Metabolism is a key component of herbicide efficiency, selectivity, and in the appearance of resistance. In many cases, oxygen transfer is the first, obligate step in the biotransformation process. These reactions are both slow and irreversible. Therefore, they determine to a large part the kinetics of elimination of the active compound (or its formation in some cases of conversion of a proherbicide to a phytotoxic compound). Although other enzymes like peroxidases may also be involved, it is now clear that cytochrome P450 plays a pivotal role in herbicide disposition. In this paper we will discuss some aspects of the biochemistry and genetics of plant P450 that are relevant to this topic, and show an example of an attempt for identifying and isolating P450 species involved in herbicide disposition.
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Research Interests: Genetics, Plant Biology, Biology, Cell Biology, Medicine, and 15 moreArabidopsis thaliana, Pseudomonas, Flagella, Plant Roots, Arabidopsis, Callose, Glucans, Pseudomonas Syringae, Plant cell, Host Pathogen Interactions, Peptidoglycan, Chitin, Biochemistry and cell biology, Jasmonic Acid, and Oxylipins
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Publisher Summary Cinnamic acid 4-hydroxylase (CA4H) catalyzes the conversion of trans-cinnamic acid to trans-4-hydroxycinnamic acid. This is the second reaction, and the first oxidative step, in the general phenylpropanoid pathway, which... more
Publisher Summary Cinnamic acid 4-hydroxylase (CA4H) catalyzes the conversion of trans-cinnamic acid to trans-4-hydroxycinnamic acid. This is the second reaction, and the first oxidative step, in the general phenylpropanoid pathway, which is common to all plants. Phenylpropanoids, and their derivatives, constitute an extremely diversified family of molecules with important biological functions or activities: precursors for lignin and suberin, pigments, aroma, defense molecules (phytoalexins), antioxidants, and UV protectants. The major difference between CA4H and most other plant P450s that are involved in species specific reactions is that CA4H is present in all plants and in almost all tissues. Assaying CA4H is therefore a way to ascertain that microsomal preparations contain intact and active P450 electron transfer chains, and provides a sort of internal standard when extraction conditions are modified. The chapter discuses (1) the nature of the enzyme source (plant species and type of tissue), (2) induction of CA4H activity, (3) preparation of microsomes, and (4) two reliable and simple assays.
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ABSTRACT
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Research Interests: Chemistry, Food Science, Medicine, Wine, Malolactic Fermentation, and 3 moreAroma, Winemaking, and Linalool
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Amaranthus retroflexus L. and Chenopodium album L. are noxious weeds that have a cosmopolitan distribution. These species successfully invade and are adapted to a wide variety of diverse climates. In this paper, we evaluated the... more
Amaranthus retroflexus L. and Chenopodium album L. are noxious weeds that have a cosmopolitan distribution. These species successfully invade and are adapted to a wide variety of diverse climates. In this paper, we evaluated the morphology and biochemistry of 16 populations of A. retroflexus L. and 17 populations of C. album L. Seeds from populations collected from Spain, France, and Iran were grown together at the experimental field of the agriculture research of University of Mohaghegh Ardabili, and a suite of morphological traits and biochemical traits were assessed. Among the populations of A. retroflexus L. and of C. album L. were observed significant differences for all the measured traits. The number of branches (BN) for A. retroflexus L. (12.22) and inflorescence length (FL; 14.34) for C. album L. were the two characteristics that exhibited the maximum coefficient of variation. Principal component analysis of these data identified four principal components for each species t...
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The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little... more
The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar ...
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SummaryMultiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long‐distance water transport. Cytochrome P450... more
SummaryMultiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long‐distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate‐limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4‐coumaroyl‐shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4‐coumaroyl‐esters and ‐a...
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Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade,... more
Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching...
Research Interests: Biochemistry, Botany, Kinetics, Biology, Medicine, and 15 moreGene Silencing, Biocatalysis, Biological Sciences, Plant Physiology, Endoplasmic Reticulum, Substrate Specificity, Quinolines, Catharanthus Roseus, Hydroxylation, PLANT PROTEINS, Indole Alkaloids, Metabolome, Biosynthetic Pathways, Plant Epidermis, and Molecular Sequence Data
Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be... more
Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process.
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Research Interests: Biochemistry, Metabolism, Biology, Biological Chemistry, Medicine, and 15 moreSignal Transduction, Biological Sciences, Plant Growth Regulators, Biological, Plant growth hormone, Arabidopsis, Enzyme, CHEMICAL SCIENCES, Genotype, Hydroxylation, Oxidation-Reduction, Catabolism, Plant Leaves, Isoleucine, and Medical and Health Sciences
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... more
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...
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The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome... more
The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different di...
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Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for... more
Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effecti...
Research Interests: Biochemistry, Biology, Medicine, Biological Sciences, Chlorophyll, and 15 moreDarkness, Light, Biochemical, Electron Transport, Heme, CHEMICAL SCIENCES, Peroxidases, Biosynthesis, Glycine, Helianthus, PLANT PROTEINS, Aminolevulinic Acid, Jerusalem artichoke, Enzyme Induction, and Medical and Health Sciences
The present invention relates to a DNA sequence which encodes a protein of interest which contains regions having a high content of codons which are poorly Suited to yeasts, characterized in that a Sufficient number of codons which are... more
The present invention relates to a DNA sequence which encodes a protein of interest which contains regions having a high content of codons which are poorly Suited to yeasts, characterized in that a Sufficient number of codons which are poorly Suited to yeasts is replaced with corresponding codons which are well-Suited to yeasts in the said regions having a high content of codons which are poorly Suited to yeasts. The present invention relates, more specifically, to DNA sequences which originate from dicotyledonous or monocotyledonous plants, in particular plants of the grami nae family which are Selected, in particular, from among Wheat, barley, oats, rice, maize, Sorghum and cane Sugar. The present invention also relates to transformed yeasts which contain a DNA sequence according to the invention.
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In plants and possibly other organisms, channelling of the reactive intermediates resulting from P450 oxygenation is thought to require the formation of supramolecular complexes associating membrane-bound and soluble enzymes. This implies... more
In plants and possibly other organisms, channelling of the reactive intermediates resulting from P450 oxygenation is thought to require the formation of supramolecular complexes associating membrane-bound and soluble enzymes. This implies a most probably loose membrane association of the soluble proteins. For the assessment of such membrane association in vivo, we propose an imaging strategy based on the accurate evaluation of fluorescent protein repartition and distance around endoplasmic reticulum (ER) tubules. It requires candidate protein fusion constructs with fluorescent reporters and transient expression in leaves of Nicotiana benthamiana. The method was tested with soluble eGFP/mRFP1, with various P450 and P450 reductase fluorescent fusions, and with anchored eGFP/mRFP1. It easily differentiated soluble and anchored proteins and detects subtle changes in ER tubules. The method was further assessed with a soluble protein previously shown to be loosely associated with the ER, the phenylalanine ammonia lyase PAL1 involved in the lignin biosynthetic pathway. This protein was found located in close vicinity to the ER. Taken together, these data indicate that the method proposed herein is suitable to monitor membrane association and relocalization of soluble proteins involved in the formation of metabolons.