Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
  • A01N37/28—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof containing the group; Thio analogues thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
  • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
  • A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the present invention relates to the use of acetohydroxyacid isomeroreductase inhibitors for the treatment of fungal diseases of cultures.
• Fungi are responsible for devastating epidemics which can cause significant losses in the cultivation of different plant species.
• the principle of employing inhibitors of enzymes of pathogenic fungi, and of using these enzymes in tests to identify new molecules active against these fungi is known per se.
• the simple characterization of a fungus enzyme is not sufficient to achieve this objective, it is still necessary that the enzyme chosen as the target of potential fungicidal molecules is essential to the life of the fungus, its inhibition by the fungicidal molecule leading to the death of the fungus, or essential to the pathogenesis of the fungus, its inhibition is not lethal for the fungus but simply inhibits its pathogenic power.
• the identification of metabolic pathways and enzymes essential to the pathogenesis and survival of the fungus is therefore necessary for the development of new fungicidal products.
• Acetohydroxyacid isomeroreductase is a well-characterized enzyme in plants and microorganisms such as bacteria and yeasts.
• This enzyme is the second enzyme in the branched chain amino acid biosynthesis pathway, it catalyzes the transformation of the substrate, 2S-2-acetolactate (AL) or 2S-2-aceto-2-hydroxybutyrate (AHB) into 2 , 3-dihydroxy-3-isovalerate (DHIN) or as 2,3-dihydroxy-3-methylvalerate (DHIM) respectively.
• This reaction requires the presence of magnesium ions (Mg 2+ ) and takes place in two stages: an isomerization of a methyl or ethyl group followed by a reduction by ⁇ ADPH.
• the present invention relates to methods of treating cultures against fungal diseases comprising the application of an acetohydroxyacid isomeroreductase inhibitor. It has been found that inactivation of the ILV5 gene encoding the acetohydroxy acid isomeroreductase in Magnaporthe grisea results in inhibition of the growth of the fungus. This inhibition of the growth of the fungus is also observed in vivo in the presence of specific inhibitors of acetohydroxyacid isomeroreductase. M. grisea is pathogenic for many crop species such as rice.
• SEQ ID No. 1 Magnaporthe grisea acetohydroxy acid isomeroreductase.
• SEQ ID No. 2 Saccharomyces cerevisiae acetohydroxyacid isomeroreductase.
• SEQ ID No. 3 Acetoohydroxy acid isomeroreductase from Neurospora crassa.
• Magnaporthe grisea SEQ ID No. 5 Magnaporthe grisea acetohydroxy acid isomeroreductase. SEQ ID No. 6 Gene of the acetohydroxy acid isomeroreductase of Magnaporthe grisea. SEQ ID No. 7-18 Primers for PCR.
• the present invention relates to methods of treating cultures against fungal diseases by applying an effective amount of an acetohydroxy acid isomeroreductase inhibitor.
• the subject of the invention is a method of combating, for curative or preventive purposes, phytopathogenic fungi in crops, characterized in that one applies to the soil where plants grow or are likely to grow, on the leaves and / or the fruits of plants or on plant seeds, an effective (agronomically effective) and non-phytotoxic amount of an acetohydroxyacid isomeroreductase inhibitor.
• effective and non-phytotoxic amount means an amount of inhibitor sufficient to allow the control or destruction of the fungi present or likely to appear on the cultures, and not causing for said cultures any significant symptom of phytotoxicity. Such an amount is likely to vary within wide limits depending on the fungus to be controlled, the type of crop, the climatic conditions, and the compounds. included in the fungicidal composition according to the invention. This quantity can be determined by systematic field tests, within the reach of those skilled in the art.
• the methods according to the invention are useful for treating cereal seeds (wheat, rye, triticale and barley in particular), potato, cotton, peas, rapeseed, corn, flax or even seeds of forest trees, or genetically modified seeds of these plants.
• the present invention also relates to the foliar application on plant crops, that is to say on the foliage, flowers, fruits and / or trunks of the plants concerned.
• rice, corn, cotton, cereals such as wheat, barley, triticale, fruit trees, in particular apple, pear, peach, vine, banana trees, orange trees, lemon trees, etc.
• oil crops for example, rapeseed, sunflower, vegetable and vegetable crops, tomatoes, salads, protein crops, peas, solaneas, for example potato, beets, flax, and forest trees, as well as genetically modified counterparts of these crops.
• plants targeted by the method according to the invention there may be mentioned:
• - wheat as regards the fight against the following seed diseases: fusarium wilt (Microdochium nivale and Fusariwn roseum), caries (Tilletia caries ,, Tilletia controversa or Tilletia indicé), septoria leaf spot (Septoria nodorum); naked coal (Ustilago tritic ⁇ ); - wheat, as regards the fight against the following diseases of the aerial parts of the plant: foot rot (Tapesia yallundae, Tapesia acuiformis), foot scald (Gaeumannomyces graminis), Fusarium head blight (F. culmorum , F.
• helminthosporioses Pyrenophora graminea., Pyrenophora teres and Cochliobolus sativus
• bare smut Ustilago nuda
• fusarium wilt Fusarium roseum
• helminthosporioses Pyrenophora graminea., Pyrenophora teres and Cochliobolus sativus
• bare smut Ustilago nuda
• fusarium wilt Fusarium roseum
• - barley as regards the fight against the following diseases of the aerial parts of the plant: foot rot (Tapesia yallundae), helminthosporioses (Pyrenophora teres and Cochliobolus sativus), powdery mildew (Erysiphe graminis forma specie horde ⁇ ), dwarf rust (Puccinia
• - cotton with regard to the fight against the following diseases of young plants grown from seed: seedlings and necrosis of the crown (Rhizoctonia solani, Fusarium oxysporum), black root rot (Thielaviopsis basicoh); - protein crops, for example peas, as regards the fight against the following seed diseases: anthracnose (Ascochyta pisi, Mycosphaerella pinodes), fusarium wilt (Fusarium oxysporum), gray mold (Botrytis cinerea), downy mildew (Peronospor ⁇ pis ⁇ );
• blast disease Magnaporthe grisea
• rhizoctonia Rostonia solan ⁇
• Acetohydroxyacid isomeroreductase is a well-characterized enzyme found in plants and microorganisms (bacteria, yeasts, fungi).
• the methods of the present invention use inhibitors of acetohydroxy acid isomeroreducatase.
• the invention relates to the use of fungus acetohydroxy acid isomeroreductase inhibitors, more preferably phytopathogenic fungal acetohydroxy acid isomeroreductase inhibitors for the treatment of fungal diseases of cultures.
• the acetohydroxyacid isomeroreductase inhibitors inhibit the acetohydroxyacid isomeroreductase of Magnaporthe grisea and / or Saccharomyces cerevisiae and / or Neurospora crassa.
• the acetohydroxyacid isomeroreductase inhibitor is an inhibitor of the enzymatic activity of acetohydroxyacid isomeroreductase from SEQ ID No. 1, from SEQ ID No.2, from SEQ ID No .3 and / or SEQ ID No. 5.
• acetohydroxyacid isomeroreductase inhibitor can be used in the methods according to the invention.
• Inhibitors of acetohydroxyacid isomeroreductase are well known to those skilled in the art and these inhibitors have in particular been described in EP106114; US4,594,098, EP196026, EP481407, WO 94/23063, CA2002021 and WO 97/37660.
• the acohydroxyacid isomeroreductase inhibitor is a reaction intermediary analog which binds to the active site of acetohydroxyacid isomeroreductase.
• the acetohydroxyacid isomeroreductase inhibitor is dimethylphosphinoyl-2-hydroxyacetate.
• the acetohydroxyacid isomeroreductase inhibitor is N-hydroxy-N-isopropyloxamate.
• the acetohydroxyacid isomeroreductase inhibitor is in the form of a fungicidal composition.
• the invention also relates to fungicidal compositions comprising an effective amount of at least one acetohydroxyacid isomeroreductase inhibitor.
• the fungicidal compositions according to the invention comprise, in addition to the inhibitor, solid or liquid supports, acceptable in agriculture and / or surfactants also acceptable in agriculture.
• the usual inert supports and the usual surfactants can be used.
• fungicidal compositions according to the invention can also contain all kinds of other ingredients such as, for example, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizers, sequestrants, etc. More generally the inhibitors of acetohydroxy acid isomero-reducatse can be combined with all the solid or liquid additives corresponding to the usual techniques of formulation.
• the present invention also relates to fungicidal compositions comprising an acetohydroxy acid isomeroreductase inhibitor and another fungicidal compound.
• Mixtures with other fungicides are particularly advantageous, in particular mixtures with acibenzolar-S-methyl, azoxystrobin, benalaxyl, benomyl, blasticidin-S, bromuconazole, captafol, captane, carbendazim, carboxin, carpropamide, chlorothalonil, fungicidal compositions based on copper or copper derivatives such as copper hydroxide or copper oxychloride, cyazofamide, cymoxanil, cyproconazole, cyprodinyl, dichloran, diclocymet, dicloran, diethofencarb, difenoconazole, diflumetorim, dimethomorph, diniconazole, discostrobin, dodemorph, do
• the present invention also relates to methods of manufacturing a fungicidal composition using an acetohydroxyacid isomeroreductase inhibitor.
• the present invention also relates to methods for the preparation of fungicidal compounds comprising the identification of compounds inhibiting the enzymatic activity of acetohydroxyacid isomeroreductase.
• the enzymatic reaction is carried out in the presence of the compound to be tested to measure the inl ibition of the enzymatic activity of acetohydroxyacid isomeroreductase.
• All the biochemical tests making it possible to measure the enzymatic activity of acetohydroxy acid isomeroreductase and therefore to identify compounds inhibiting this enzymatic activity can be used in the methods according to the invention.
• These biochemical tests are well known to those skilled in the art (Dumas et al., Biochem. J. 288: 865-874, 1992; Dumas et al., Biochem. J. 301: 813-820, 1994; Dumas et al.
• the enzymatic reactions are advantageously carried out in solution in an appropriate buffer.
• This type of reaction medium makes it possible to carry out a large number of reactions in parallel and therefore to test a large number of compounds in a microplate format for example.
• the methods for identifying compounds inhibiting the enzymatic activity of acetohydroxy acid isomeroreductase comprise contacting these compounds with acetohydroxy acid isomeroreductase in the presence of magnesium, of NADPH and of substrate; and measuring this enzymatic activity.
• the measurement of the enzymatic activity comprises the measurement of the decrease in absorption of NADPH at 340 nm and the substrate used for the enzymatic reaction is 2S-2-acetolactate (AL) or 2S-2-aceto-2-hydroxybutyrate (AHB). It is understood that any other method of measuring activity enzyme known to the skilled person can be used in the methods according to the invention.
• Acetohydroxy acid isomeroreductase can be used in the methods according to the invention.
• Acetohydroxyacid isomeroreductases have been characterized in several organisms such as plants, bacteria, yeasts and fungi. The corresponding genes have been cloned to determine the protein sequence of this enzyme (Dumas et al, Biochem. J. 277: 69-475, 1991; Curien et al, Plant Mol. Biol. 21: 717-
• the acetohydroxy acid isomeroreductase used in the methods according to the invention is represented in SEQ ID No. 1, in SEQ ID No.2, in SEQ ID No.3 and / or to SEQ ID No.5.
• the acetohydroxy acid isomeroreductase is isolated, purified or partially purified from its natural environment.
• Acetohydroxyacid isomeroreductase can be prepared by various methods. These methods include purification from natural sources such as cells naturally expressing these polypeptides, production of recombinant polypeptides by appropriate host cells and their subsequent purification, production by chemical synthesis or, finally, a combination of these different approaches. . These various production methods are well known to those skilled in the art.
• the acetohydroxy acid isomeroreductase is purified from an organism naturally producing this enzyme, for example bacteria such as E. coli, yeasts such as S. cerevisiae, or fungi such than N.crassa or M. grisea.
• the acetohydroxy acid isomeroreductase is overexpressed in a recombinant host organism.
• the methods of engineering DNA fragments and the expression of polypeptides in host cells are well known to those skilled in the art and have for example been described in "Current Protocols in Molecular Biology” Volumes 1 and 2, Ausubel FM et al, published by Greene Publishing Associates and Wiley-Interscience (1989) or in Molecular Cloning, T. Maniatis, EFFritsch, J. Sambrook (1982).
• the methods for identifying compounds which inhibit the enzymatic activity of acohydroxyacid isomeroreductase comprise the expression of acetohydroxyacid isomeroreductase in a host organism, the purification of acetohydroxyacid isomeroreductase produced by the host organism, contact of these compounds with purified acetohydroxyacid isomeroreductase in the presence of magnesium, NADPH and substrate; and measurement of enzyme activity.
• all of these methods include an additional step in which it is determined whether said compounds inhibiting the enzymatic activity of acetohydroxy acid isomeroreductase inhibit the growth and / or pathogenesis of fungi.
• the present invention therefore relates to methods for identifying compounds which inhibit the growth and / or pathogenesis of fungi by inhibiting the enzymatic activity of acetohydroxyacid isomeroreductase. These methods consist in subjecting a compound, or a mixture of compounds, to an appropriate test for the identification of the acetohydroxyacid isomeroreductase inhibitor compounds and to selecting the compounds reacting positively to said test, if necessary to isolating them, then to identify them.
• the appropriate test is a test of the enzymatic activity of acetohydroxyacid isomeroreductase as defined above.
• a compound identified according to these methods is then tested for these anti-fungal properties and for its capacity to inhibit the pathogenesis and / or the growth of the fungus for plants according to methods known to those skilled in the art.
• the compound is evaluated using phenotypic tests such as pathogenesis tests on leaves or on whole plants.
• phenotypic tests such as pathogenesis tests on leaves or on whole plants.
• compound is meant according to the invention any chemical compound or mixture of chemical compounds, including peptides and proteins.
• mixture of compounds is understood according to the invention at least two different compounds, such as for example the (dia) stereoisomers of a molecule, mixtures of natural origin resulting from the extraction of biological material (plants, plant tissues, culture bacteria, yeast or fungal cultures, insects, animal tissues, etc.) or unpurified reaction mixtures or wholly or partly purified, or mixtures of products from combinatorial chemistry techniques.
• biological material plants, plant tissues, culture bacteria, yeast or fungal cultures, insects, animal tissues, etc.
• unpurified reaction mixtures or wholly or partly purified, or mixtures of products from combinatorial chemistry techniques.
• the present invention finally relates to new compounds which inhibit the pathogenesis of fungi inhibiting the enzymatic activity of acetohydroxy acid. isomeroreductase, in particular the compounds identified by the methods according to the invention and / or the compounds derived from the compounds identified by the methods according to the invention.
• the compounds inhibiting the pathogenesis of fungi inhibiting the enzymatic activity of acetohydroxyacid isomeroreductase are not general inhibitors of enzymes.
• the compounds according to the invention are not compounds already known to have fungicidal activity and / or activity on the pathogenesis of fungi.
• the subject of the invention is also a method for treating plants against a phytopathogenic fungus, characterized in that it comprises the treatment of said plants with a compound identified by a method according to the invention.
• the present invention also relates to a method for preparing a compound inhibiting the pathogenesis of fungi, said method comprising the steps of identifying a compound inhibiting the pathogenesis of fungi inhibiting the enzymatic activity of acetohydroxy acid isomeroreductase by the method identification according to the invention, then preparation of said compound identified by the usual methods of chemical synthesis, enzymatic synthesis and / or extraction of biological material.
• step of preparation of the compound can be preceded if necessary by a step called optimization by which a compound derived from the compound identified by the identification process according to the invention is identified, said derivative compound then being prepared by the methods usual.
• Figure 1 Comparison of the protein sequences of the isomeroreductases of M. grisea, N.crassa and of the yeast S. cerevisiae. Alignment of sequences using CLUSTAL W software (1.4). .: similar amino acid.
• FIG. 1 Growth test of M. grisea in the presence of the inhibitor N-hydroxy-N-isopropyloxamate (IpOHA) and under different culture conditions.
• IpOHA N-hydroxy-N-isopropyloxamate
• the effect of the inhibitor IpOHA is tested on the pathogenic fungus M. grisea by following the evolution of the growth of this fungus in the presence of different concentrations of inhibitor, in different culture media and over a period of 7 days.
• PI .2 of M grisea at a final concentration of 10 5 spores / ml.
• the microplate is incubated at room temperature and the optical density at 630 nm (OD 63 o) is measured on days O, 3,
• Figure 3 Influence of the concentration of NADPH on the enzymatic activity of the yeast isomeroreductase.
• the enzymatic activity measurements are carried out at 25 ° C. in
• the K M for NADPH is 1.6 ⁇ M.
• Figure 4 Influence of the concentration of substrate AHB [A] and AL [B] on the enzymatic activity of the yeast isomeroreductase.
• the enzymatic activity measurements are carried out at 25 ° C. in the following reaction medium: MgCh 10 mM, NADPH 250 ⁇ M and in 1 ml of Hepes sodium buffer 50 mM, pH 7.5 and in the presence of AHB [A] and AL
• a The K M for AHB is 104 ⁇ M.
• B The K M for LA is 266 ⁇ M.
• Figure 5 Influence of the magnesium concentration on the enzymatic activity of the yeast isomeroreductase.
• the enzymatic activity measurements are carried out at 25 ° C. in 1 ml of the following reaction medium: MgCte from 0 to 40 mM, NADPH 250 ⁇ M, AHB 0.48 mM and in the 50 mM sodium Hepes buffer, pH 7.5.
• the curve is fitted to the Michaelis Menten model.
• the K M for Mg 2+ is 968 ⁇ M.
• Figure 6 Stoichiometry of binding of the inhibitors Dimethylphosphinoyl-2-hydroxyacetate and N-hydroxy-N-isopropyloxamate on the yeast isomeroreductase.
• the inhibitors Hoe 704 [A] and IpOHA [B] (0.1 nmoles) are incubated with variable amounts of enzyme (0 to 0.4 nmoles) for 20 minutes at 25 ° C with 25 nmoles of NADPH and 0.25 ⁇ moles of Mg 2+ in a volume of 10 ⁇ l.
• the enzymatic activity measurements are carried out at 25 ° C. in 1 ml of the following reaction medium: MgCh 10 mM, NADPH 250 ⁇ M, AHB 0.48 mM and in the Hepes sodium buffer 50 mM, pH 7.5.
• Figure 7 Kinetics of inhibition of yeast isomeroreductase in the presence of inhibitors dimethylphosphinoyl-2-hydroxyacetate [A] and N-hydroxy-N-isopropyloxamate [B].
• the enzymatic activity measurements are carried out at 25 ° C. in 1 ml of the following reaction medium: MgC 10 mM, NADPH 250 ⁇ M, AHB 0.48 mM in the Hepes sodium buffer 50 mM, pH 7.5 and in the presence of enzyme at 110 nM.
• the reactions are initiated by simultaneously adding variable amounts of inhibitors dimethylphosphinoyl-2-hydroxyacetate [A] and N-hydroxy-N-isopropyloxamate [B] (from 2 ⁇ M to 30 ⁇ M) in the reaction medium.
• the curves are plotted according to equation (1), which allows the determination of the values of K 0bs , apparent speed of formation of the enzyme-inhibitor complex.
• Figure 8 Kinetics of inhibition of yeast isomeroreductase in the presence of inhibitors dimethylphosphinoyl-2-hydroxyacetate [A] and N-hydroxy-N-isopropyloxamate
• the enzymatic activity measurements are carried out at 25 ° C. in 1 ml of the following reaction medium: MgCk 10 mM; NADPH 250 ⁇ M; 0.48 mM AHB in 50 mM sodium Hepes buffer, pH 7.5 and in the presence of an enzyme at 110 nM.
• the reactions are initiated by simultaneously adding variable amounts of inhibitors [A] and [B] (from 2 ⁇ M to 50 ⁇ M) in the reaction medium.
• Figure 9 Determination of the association constant k 0 of dimethylphosphinoyl-2-hydroxyacetate [A] and N-hydroxy-N-isopropyloxamate [B] on the yeast isomeroreductase thanks to the representation 1 / Kobs as a function of the concentration of AHB substrate.
• the enzymatic activity measurements are carried out at 25 ° C. in 1 ml of the following reactive medium: MgCh 10 mM, NADPH 250 ⁇ M, AHB from 125 ⁇ M to 2.375 mM and in the Hepes sodium buffer 50 mM, pH 7.5 and in the presence of enzyme at 110 nM.
• the reactions are initiated by simultaneously adding variable amounts of AHB substrate (from 125 ⁇ M to 2,375 mM) and the inhibitors [A] (10 ⁇ M) and [B] (15 ⁇ M) in the reaction medium.
• An internal fragment of the M. Grisea ILV5 gene was amplified by PCR from the genomic DNA of this fungus using pairs of degenerate primers corresponding to protein domains conserved between the isomeroreductases of fungi.
• the PCR product obtained was then cloned into the plasmid pGEM-T-Easy (Promega), sequenced and amplified by PCR with a new pair of primers.
• the latter PCR product was used as a homologous probe to screen a M. grisea cosmid DNA library.
• the M. grisea ILV5 gene sequence was then produced from of one of the positive clones and of oligonucleotides derived from the sequence of the PCR product already obtained.
• Amplification of an internal fragment of the M. Grisea ILV5 gene was carried out by PCR using degenerate oligonucleotides. These degenerate oligonucleotides were chosen from the comparison of the protein sequences of the isomeroreductases of N. crassa and S. cerevisiae. This comparison made it possible to highlight 4 domains conserved between these two sequences of fungal isomeroreductases, which should be present in the isomeroreductase of M. grisea. These 4 conserved domains consist of a succession of at least 7 conserved amino acids. The sequence of degenerate oligonucleotides was determined from that of the conserved domain amino acids translated according to the genetic code.
• PCR amplification can be carried out with four different pairs of degenerate oligonucleotides: 1 (+) and 2 (-); 1 (+) and 4 (-); 3 (+) and 2 (-); 3 (+) and 4 (-). 1.1.2. Amplification of an internal fragment of the M. Grisea ILV5 gene using degenerate oligonucleotides
• the optimal conditions for amplification of the M. Grisea ILV5 gene were determined by varying the pair of primers and their hybridization temperature.
• the first 3 amplification cycles were carried out at a variable hybridization temperature (42 ° C, 50 ° C or 55 ° C), while the hybridization temperature of the other cycles was 55 ° C.
• Positive controls were carried out with the genomic DNA of N. crassa and of S. cerevisiae under the same conditions as for the genomic AD ⁇ of M. grisea. At a hybridization temperature of 42 ° C., the DNA fragments of S.
• crassa were amplified to the expected size, namely 660 bp, 685 bp, 523 bp and 544 bp with the pairs of primers (1-4) , (1-2), (3-4) and (3-2), respectively, although the amplification profiles with the pairs of primers (1-4) and (3-4) are complex.
• the different pairs of degenerate oligonucleotides made it possible to amplify, from the genomic AD ⁇ of yeast and of N. crassa, a fragment of expected size. These degenerate oligonucleotides could therefore be used to amplify the M. grisea ILV5 gene.
• the quantity of oligonucleotides used, tested at a hybridization temperature of 42 ° C., does not seem to have any influence on the amplification of the genomic DNA of M. grisea.
• the amplification profiles of the genomic DNA of M. grisea, obtained with the different pairs of primers are quite complex.
• a simplification of the amplification profiles of the genomic DNA of M. grisea was observed for most of the primer pairs; in particular for the pair of primers (1 - 2) which makes it possible to amplify a single DNA fragment to the expected size (685 bp).
• the internal fragment of the M. Grisea ILV5 gene was amplified by PCR from the genomic DNA of M. grisea, with the pair of primers (1-2), at a primer hybridization temperature of 50 °. C for 3 cycles, then 55 ° C for the other amplification cycles.
• This PCR product of about 680 bp, is purified after separation by agarose gel electrophoresis and then cloned into the plasmid pGEM-T-easy.
• the bacterial colonies obtained after transformation and thanks to a white / blue selection system (X-Gal) showed three different phenotypes: white, blue and white with the center of the blue colony (called white / blue colonies).
• nucleotide sequences of the two clones # 4 and # 20 has shown that they correspond to the same DNA fragment cloned in different orientations in the plasmid pGEM-T-easy, which could explain their phenotypic difference ( white and white / blue).
• the double-stranded nucleotide sequence of this cloned fragment was thus obtained.
• the homology of this nucleotide sequence with those coding for known proteins was sought thanks to the Blastx program of NCBI (National Center for Biotechnology Information). This program compares the translated sequences of the six reading phases of a nucleotide sequence with all of the protein sequences contained in the databases.
• the fragment cloned into the plasmid pGEM-T-easy therefore corresponds to the internal fragment of the ILV5 gene from M. grisea. Although the sequence of the internal fragment of the ILV5 gene of M. grisea and that of the ILV5 gene of N. crassa show strong homology, a difference at the center of the sequence of the ILV5 gene of M. grisea exists.
• This difference could correspond to the presence of an intron within the sequence of M. grisea, since an intron of 77 bp exists in N. crassa at this position. The position and the sequence of this intron were sought.
• a 5 ′ consensus splicing motif was identified in the nucleotide sequence of the internal fragment of the M. Grisea ILV5 gene, as well as a 3 ′ consensus splicing motif and a lariat sequence.
• the putative intron (86 bp) of the internal fragment of the M. Grisea ILV5 gene was therefore identified. Splicing the intron of the sequence of the internal fragment of the ILV5 gene of M. grisea makes it possible to obtain a “theoretical” fragment of AD ⁇ c.
• An internal fragment of the M. Grisea ILV5 gene was amplified by PCR, from clone No. 4, using primers 13U and 549L defined from the sequence of the ILV5 gene cloned in the plasmid pGEM-T- easy. This fragment, after purification on agarose gel, was used as a template to produce a labeled ILV5 gene probe.
• the Guyl 1 cosmid library is represented in the form of 28 pools of 96 DNA mini-preparations corresponding to the 96 different cosmids present in a 96-well plate (2,688 clones).
• the ILV5 gene of M. grisea was sought in this cosmid library by carrying out a PCR amplification on these pools of DNA mini-preparations using the primers 300U and 549L defined from the known sequence of the ILV5 gene.
• a fragment of expected size (249 bp) was amplified from pools No. 17; 19; 20; 21; 27; 28 and 29.
• the search for the ILV5 gene was continued by hybridizing with the ILV5 gene probe the cosmids of plates No. 17; 19; 20; 21; 27; 28 and 29.
• the sequencing of the ILV5 gene was carried out in stages.
• the first sequence reaction was carried out using divergent primers chosen from the known sequence of the internal fragment of the ILV5 gene from M. grisea obtained by PCR. From this new sequence of the ILV5 gene, new primers have been defined to carry out other sequence reactions and this until the ILV5 gene of M. grisea has been completely sequenced.
• the sequences translated from the entire nucleotide sequence of the ILV5 gene according to the 6 reading phases are compared with the protein sequence of the N. crassa isomeroreductase, in order to locate the position of the translation initiating ATG, of the STOP codon. of termination of the translation, and of the different introns possible.
• the translation initiating ATG has been identified on the nucleotide sequence of the ILV5 gene and serves as a reference (+1) from which the other elements of the sequence are positioned.
• Three putative mirons have been identified in the nucleotide sequence of the M. grisea ILV5 gene. The first intron would be located between positions (in bp) 199 and 280, the second intron in position 314-390 and the third intron would be located between positions 670 and 755 of the ILV5 gene of M. grisea.
• the translation termination STOP codon would be at position 1449 of the M. grisea ILV5 gene sequence. The isolation of the M.
• Grisea ILV5 gene cDNA was undertaken in order to verify the position of the suspected introns by comparing the protein sequences of M. grisea and N. crassa. b) Isolation of AD ⁇ c from the ILV5 gene of M.grisea and search for introns of the gene of M.grisea
• the AD ⁇ c of the ILV5 gene of M. grisea was isolated by carrying out a PCR amplification from an AD ⁇ c library of the isolate PI.2 (AR ⁇ of mycelium grown in complete medium) using the defined oligonucleotides from the ILV5 gene sequence: oligonucleotides 22U and 1603L.
• the oligonucleotide 22U is located before the translation initiating ATG and the oligonucleotide 1603L is located 93 bp after the STOP codon for terminating the translation.
• Two fragments are amplified with this pair of primers: a fragment of size less than 500 bp and a fragment amplified to the expected size, ie 1.6 kb.
• the fragment amplified to the expected size is purified after separation by agarose gel electrophoresis and cloned into the plasmid pGEM-T-easy.
• the 24 bacterial colonies obtained after transformation are analyzed by PCR using the pair of primers 22U and 1603L. These 24 clones have the AD ⁇ c of the ILV5 gene of M. grisea, because a DNA fragment has indeed been amplified to the expected size (1.6 kb).
• Clone # 18 was chosen to be sequenced using the universal primers Sp6 and T7.
• the three introns are located at the positions predicted by the comparison of the protein sequences of the isomeroreductases of N. crassa and translations of the ILV5 gene of M. grisea.
• the ILV5 gene has 4 introns positioned differently and of different lengths compared to the IL V5 gene of M. grisea.
• the protein sequence of the M. Grisea ILV5 gene has been deduced from the T AD ⁇ c sequence of this gene. Comparison of the protein sequences of the isomeroreductases of M. grisea, N. crassa and S. cerevisiae show a very strong identity between the isomeroreductases of these three species. Indeed, the percentage of identity between the sequences of the isomeroreductases of M. grisea and N. crassa is 86%. That between the isomeroreductases of M. grisea and yeast is 70% and that between the isomeroreductases of N. crassa and yeast is 72% ( Figure 1). N. crassa and M.
• grisea are very similar fungal species (pyrenomycetes), which could explain the high identity percentage between the isomeroreductases of these two species. d) Study of the expression of the isomeroreductase of M.grisea in this fungus subjected to different stress conditions The total AR ⁇ s of M. grisea coming from mycelium subjected to different stress conditions were extracted and then transferred to the membrane before be hybrid with the homologous probe of the M. grisea ILV5 gene. The ILV5 gene is expressed constitutively. It is thus expressed at the same level during a hyperosmotic stress or a nitrogenous nutritional deficiency or an induction by cAMP, a thermal shock or an oxidative stress. It is however not expressed during a carbon nutritional deficiency.
• the ILV5 gene is subcloned in the plasmid pBC SK + before insertional mutagenesis by transposon.
• Cosmid 20 / G6 containing the ILV5 gene carries an ampicillin resistance gene, so it cannot be used directly as a target for insertional mutagenesis. Indeed, a double selection by kanamycin and ampicillin would not be selective enough for the target plasmids which have integrated the transposon, since the transposon donor plasmid (pGPS3 Hygro) is also resistant to kanamycin and to ampicillin.
• the ILV5 gene was therefore subcloned into a plasmid carrying a chloramphenicol resistance gene, the plasmid pBC SK +.
• the clones having integrated the transposon at the level of the target plasmid may be selected for their double resistance to kanamycin and to chloramphenicol.
• the size of the cosmid insert is too large (40 kb).
• the length of the ILV5 gene is approximately 3 kb, the probability that the transposon integrates into the gene present at the cosmid level (46 kb) is 6.5%, while the probability of integration of the transposon into the ILV5 gene subcloned into the plasmid pBC SK + (18 kb) is approximately 3 times higher, or approximately 17%.
• the subcloning of the genomic DNA fragment carrying the ILV5 gene was carried out by positioning this gene at the center of the insert. This type of construction facilitates the integration of the mutated ILV5 gene into the genome of M. grisea by homologous recombination.
• the mapping of the cosmid 20 / G6 region carrying ILV5 made it possible to choose a 15 kb Clal-Clal fragment in which the ILV5 gene is relatively well centered. Indeed, the ILV5 gene is framed in 5 'by 5 kb of the genomic sequence and in 3' by 5.5 kb.
• the 15 kb fragment containing the ILV5 gene was purified after separation on an agarose gel and under clone in the plasmid pBC SK +. 24 colonies obtained after transformation were analyzed by PCR using the pair of primers 22U and 1603L specific for the ILV5 gene.
• the 5 clones amplify a fragment to the expected size (1.6 kb) possessing the ILV5 gene.
• Clone No. 19 was chosen to carry out insertional mutagenesis by in vitro transposition of the IL V5 gene.
• Insertional mutagenesis by in vitro transposition of the ILV5 gene from M. grisea was carried out with the GPS T kit (New England Biolabs) from clone No. 19.
• the plasmid pGPS 3 Hygro which carries a gene for resistance to kanamycin and to hygromycin at the level of the transpressor, is used as a transposon donor.
• the plasmid pBC SK + carrying the ILV5 gene and the chloramphenicol resistance gene, corresponds to the target plasmid.
• the bacterial clones having an integration of the transposon at the level of the target plasmid pBC SK + are selected by virtue of their resistance to both kanamycin and chloramphenicol. This double resistance could also be conferred on bacterial clones having both the target plasmid and the donor plasmid intact. Destruction of the donor plasmid by digestion with the enzyme Pl-Scel overcomes this problem.
• the selected bacterial clones are analyzed by PCR using the pair of primers 22U and 1603L, located on either side of the coding region of the ILV5 gene.
• Bacterial clones # 3; 8; 18 and 29 were sequenced using divergent primers Tn7L and Tn7R located at each end of the transposon, in order to locate the exact position of the site of insertion of the transposon Tn7 within the sequence of the ILV5 gene.
• the transposon integrated 21 bp before ATG for clone # 18, 9 bp after ATG for clone # 8, 809 bp after ATG for clone # 3 and 1176 bp after ATG for clone # 29.
• the insert of clone No. 8 containing the ILV5 gene of M. grisea disrupted in its coding region (9 bp after ATG) emerged from the plasmid by digestion with the enzyme CM and purified on agarose gel. It corresponds to the linearized construction.
• the plasmid pBC SK + from the undigested clone No. 8 corresponds to the "circular" construction. Transformation of the protoplasts of the PI.2 strain. of M. grisea is carried out either with 5 ⁇ g of the linearized construction, or with 4 ⁇ g of the "circular" construction.
• the positive transformation control is carried out using 3 ⁇ g of plasmid pCB1003 carrying a hygromycin resistance gene and the negative control is carried out without DNA.
• 62 transformants are obtained for linearized construction and 24 for "circular" construction.
• These 86 transformants were subcultured on complete medium supplemented with hygromycin at 120 mg / 1 and on minimum medium supplemented with hygromycin at 120 mg / 1 or at 60 mg / 1.
• This type of subculture makes it possible to identify the transformers ilv5 ⁇ which are auxotrophic for leucine, valine and isoleucine and therefore which do not grow on minimum medium.
• auxotrophic transformants are carried out by subculturing these colonies from these monospores on minimum medium, on minimum medium supplemented with leucine, valine and isoleucine at 0.3 mM and on complete TNKYE glucose medium. Thus, genetically purified and stable transformants were obtained. Out of 10 potential auxotrophic transformants for leucine, valine and isoleucine, 8 (80%) were found to be effectively auxotrophic. The 2 non-auxotrophic transformants had to correspond to a mixture of genetically different populations (ilv5 and ilv5 " ) which evolved towards a majority of ilv5 during the growth of the transformant on non-selective medium before monospore purification.
• Example 3 Phenotic characterization of the ilv5 ⁇ transformants of auxotrophic Magnaporthe grisea for leucine, valine and isoleucine and study of their pathogenic power 3.1. Effect of disruption of the ILV5 gene on the growth and development of M.grisea transformants The development of the ilv5 transformants " was tested on different culture media. Thus, on minimum nitrate medium, the ilv5 " transformants are unable to grow, while their growth is possible on minimum medium supplemented with valine, leucine and isoleucine at 0.3 mM.
• the development of the ilv5 " transformants on minimum medium supplemented with 0.3 mM valine and isoleucine can be improved by supplementing the minimum medium with a final concentration of valine and isoleucine of 1 mM.
• the ilv5 " transformants exhibit little different from the wild strain: their mycelium is gray-white more or less aerial (less aerial than the wild strain).
• adding of panthoteine, oxidized form of panthotenate which is involved in the biosynthesis of leucine, at 1 mg / 1 in the minimum medium + valine and isoleucine at 0.3 mM does not improve the development of the transformants ilv5 " .
• the sporulation of transformants ilv5 " is more slow and ten times less important on agar medium "rice flour” compared to the wild strain.
• the sporulation of the ilv5 " transformants is almost identical to the wild strain when valine and isoleucine are added to the" rice flour "agar medium at a final concentration of 1 mM.
• M. grisea pathogenicity tests of ilv5 " transformants were carried out by brushing or depositing drops of spore suspension of the transformants to be tested on cut barley leaves.
• An inoculation is carried out using a wet cotton swab dipped in a suspension of spores (3.10 ⁇ spores / ml in general) and used to brush the fragments of barley leaves in survival (on medium 1% agar water, kinetin 2 mg / 1).
• the other type of inoculation consists of depositing 30 ⁇ l drops in three different places on the surface of the barley leaves. Symptoms are observed after 5 to 9 days of incubation at 26 ° C.
• the lesions caused by the ilv5 " mutants are also atypical and appear later. They appear after 6 to 9 days of incubation at 26 ° C., compared with 4 to 9 days for the wild strain (see table 2 below). Some lesions caused by transformants ilv5 " appear at the ends of the barley leaves (Table 2). Injuries at the tips of the leaves could explain these lesions, as they could facilitate penetration of the fungus. Pathogenicity tests on whole plants have therefore been carried out, in order on the one hand to confirm the existence of a reduction in the pathogenic power of the transformants ilv5 " and on the other hand to estimate the reduction in the pathogenesis of the mutants ilv5 " and the importance of injuries for the penetration of the ilv5 fungus " .
• the pathogenicity tests of ilv5 transformants " were carried out by spraying a suspension of spores (10 and 3.10 spores.ml " ) from these barley plants. Gelatin at a final concentration of 0.5% (w / v) is added to this suspension of spores to allow better adhesion of the spores to the surface of the leaves. The plants are placed in a humid room overnight after inoculation. Symptoms are usually seen after 5-10 days of room temperature incubation for barley.
• Table 3 Test of pathogenic power of transformants ilv5 " on whole plant (barley) The inoculation of barley plants was carried out by spraying a suspension of spores
• the fungicidal effect of the dimethylphosphinoyl-2-hydroxyacetate and N-hydroxy-N-isopropyloxamate inhibitors was tested on the pathogenic fungus M. grisea by following the evolution of the growth of this fungus in the presence of different concentrations of inhibitor, over a period of 7 days.
• a dom ed culture medium minimum nitrate medium (MM); MM + leucine, valine and isoleucine at 0.3 mM; complete TNKYE glucose medium (MC); MC + leucine, valine and isoleucine at 0.3 mM
• MM minimum nitrate medium
• MC complete TNKYE glucose medium
• N-hydroxy-N-isopropyloxamate at concentrations varying from 0.03 ⁇ M to 3 ⁇ M
• different media MM and MM supplemented with leucine, valine and isoleucine at 0.3 mM.
• N-hydroxy-N-isopropyloxamate strongly inhibits the growth of M. grisea. This inhibition of growth by N-hydroxy-N isopropyloxamate (from 0.3 .mu.M) is observed from the 3rd day of growth to me, and like the rest following days ( Figure 2).
• the effect of the inhibitor N-hydroxy-N-isopropyloxamate was tested on the pathogenic fungus M. grisea by following the evolution of the growth of this fungus in the presence of different concentrations of inhibitor, in different culture media and on a period of 7 days.
• the values given in the table correspond to the average growth percentages of the M. grisea fungus obtained on the 5th day of growth from two tests.
• the control wild strain of M. grisea corresponds to a growth rate of 100%.
• N-hydroxy-N-isopropyloxamate By supplementing the minimum medium with valine, leucine and isoleucine to 0.3 mM, the toxicity of N-hydroxy-N-isopropyloxamate on the growth of the fungus M. grisea is raised, whatever the concentration of N-hydroxy-N-isopropyloxamate used.
• This lifting of toxicity of N-hydroxy-N-isopropyloxamate by the addition of valine, leucine and isoleucine to the minimum medium shows that this toxicity results from a specific inhibition of the pathway for biosynthesis of these amino acids, by inhibiting isomeroreductase.
• N-hydroxy-N-isopropyloxamate acting specifically on the isomeroreductase of M. grisea strongly inhibits the growth of M. grisea at very low concentrations.
• the isomeroreductase and the inhibitor N-hydroxy-N-isopropyloxamate appear to be a good target
• N-hydroxy-N-isopropyloxamate had an effect on the germination of M. grisea spores.
• Additional tests were performed to determine if N-hydroxy-N-isopropyloxamate at high concentrations could block spore germination. Thus, at 10 mM, N-hydroxy-N-isopropyloxamate does not inhibit the germination of M.
• DI 50 concentration inhibiting the growth of the fungus by 50%.
• DI 80 concentration inhibiting the growth of the fungus by 80%.
• the coding sequence of the yeast ILV5 gene without the transit peptide was overexpressed in E. coli in order to obtain large quantities of enzyme to facilitate its biochemical study and in particular its structural study.
• yeast isomeroreductase gene ILV5 was first amplified by PCR without the part coding for the transit peptide. The PCR reaction product was then cloned into a pTP expression vector inducible by 1TPTG. The isomeroreductase was then overproduced in E. coli, purified and its biochemical properties studied.
• the signal peptide which allows the yeast isomeroreductase to be addressed in its cell compartment, the mitochondria, is cleaved when the protein has entered the mitochondria.
• the region of the yeast ILV5 gene located between the end of the transit peptide and the translation termination STOP codon was amplified by PCR from the genomic DNA of S. cerevisiae with the pairs of primers (l'-3 ') and (2'-3').
• the size of the DNA fragments amplified with the pairs of primers (1'-3 ') and (2'-3') is 1079 bp and 1124 bp, respectively.
• Double digestion of the cloning vector and of the PCR reaction product with the enzymes Ncol and Sali makes it possible in fact to clone the yeast ILV5 gene in the correct orientation in the vector PET-23d.
• the plasmid pET-23d (Tebu) carrying the ampicillin resistance gene, is used as an inducible expression vector to produce a large quantity of yeast isomeroreductase in E. coli.
• This type of vector has a promoter of phage T7, which is recognized by T7 RNA polymerase, but not by E.co / z TARN polymerase.
• the production of the cloned protein takes place after the induction by 1TPTG of the strain BL21 pLysS
• the T7 RNA polymerase gene is under the control of the lac promoter inducible by 1TPTG.
• the BL21 pLysS strain transformed with the plasmid p ⁇ T-23d, carrying the ILN5 gene from S. cerevisiae is called BL21 pLysS-p ⁇ T-23d-isomeroreductase.
• Construction No. 1 corresponds to the cloning of the amplified fragment with the primers (1 '-3') and Construction No. 2 corresponds to the cloning of the amplified DNA fragment with the primers (2 '-3').
• a double Sall / Ncol digestion of the 12 clones obtained after transformation of the DH5 cells with construction no. 1 and of the 6 clones transformed with construction no. 2 makes it possible to bring out the cloned fragment in the vector p ⁇ T and thus to verify the presence of one of the 2 constructions within the different clones. Analysis of the digestion profiles of these bacterial clones showed that they all had the corresponding construction.
• Two clones were selected: the clone P ⁇ T 1-4 (construction n ° 1) and the clone P ⁇ T 2-1 (construction n ° 2); they were used to transform BL21 pLysS cells, in order to overproduce the yeast isomeroreductase in E. coli.
• the overexpression of the "short" form (construction No. 1) and the “long” form (construction No. 2) of the yeast isomeroreductase was induced in E. coli by 1TPTG.
• the bacterial strain BL21 pLysS, transformed with the plasmid p ⁇ T 23-d containing the yeast ILV5 gene without the region coding for the transit peptide, is cultured at 28 ° C. with stirring in LB medium supplemented with carbenicillin (100 mg / 1) and in chloramphenicol (30 mg / 1) up to a density equivalent to an OD 600 of approximately 0.6.
• LTPTG is then added to a concentration of 0.4 mM final and the bacteria are left in culture at 28 ° C. with stirring for approximately 15 hours.
• This bacterial culture is then centrifuged (30 minutes, 4500 rpm); the bacterial pellet is resuspended in 15 ml of buffer (KH2-K2PO4 10 mM (pH 7.5), ⁇ DTA 1 mM, DTT 1 mM and protease inhibitors: benzamidine HC1 1 mM, amino-caproic acid 5 mM) and sonicated by Vibra - cell disruptor (Sonics and Materials, Danbury, CT, US A) for 15 minutes, at power 4.40% of the total lysis time.
• the cell extract is centrifuged (20 minutes, 15,000 rpm); the supernatant, containing the soluble proteins, is then stored at -80 ° C.
• the purification of the short form of isomeroreductase was therefore carried out in two stages starting from the fraction of soluble proteins; first on an anion exchange column (Q-Sepharose), then on a permeation column (Superdex 75).
• the soluble protein extract (15.5 ml; 227.8 mg protein), which contains the yeast isomeroreductase (crude extract) is applied to an anion exchange column, HiLoad 16/10 Q- Sepharose (Pharmacia) connected to a Pharmacia FPLC system, previously equilibrated with 10 mM KH2-K2PO4 buffer / 1 mM EDTA / 1 mM DTT.
• the chromatographic fractions containing the yeast isomeroreductase are concentrated to 1.6 ml by centrifugation at 5500 rpm in a macrosep-10 unit (filter).
• This extract (27.7 mg) is then applied to a Hiload 16/60 Superdex 75 column (Pharmacia) connected to an F.P.L.C system from Pharmacia, previously equilibrated with Hepes KOH 25 mM buffer. .
• the chromatographic fractions containing the yeast isomeroreductase (18.99 mg) are concentrated to 9.7 mg / ml by centrifugation at 5500 rpm in a 10K microsep (filter) and stored at -80 ° C.
• the yeast isomeroreductase After injection of the soluble protein fraction (approximately 230 mg) into the Q-Sepharose column, the yeast isomeroreductase is eluted with 10 mM KH2-K2PO4 buffer / 1 mM EDTA / 1 mM DTT. It is in fact useless to elute this enzyme by acting a gradient of increasing concentration of phosphate buffer since preliminary experiments have shown that this enzyme is not retained by the column. After this first purification step, approximately 30 mg of protein was recovered and the purification yield in terms of activity is 55% (Table 6). Table 6: Steps in the purification of the overexpressed yeast isomeroreductase in E. coli
• the activities were determined in the following reaction medium: Hepes of sodium 50 mM, pH 7.5; MgCl 2 10 mM; NADPH 250 ⁇ M and AHB 0.48mM.
• the determination of the kinetic parameters of the yeast isomeroreductase was carried out by monitoring the evolution of the enzymatic reaction using a spectrophotometer under saturated conditions of magnesium, NADPH and substrate AHB or AL. All the measurements of enzymatic activity were carried out in a quartz cell with a 1 cm optical path containing sodium Hepes buffer (50 mM, pH 7.5), 10 mM MgCl 2 , 250 ⁇ M NADPH, in a volume final 1 ml, at 25 ° C. The enzymatic reaction was initiated by the addition of 0.48 mM of AHB or AL. The progress of this reaction was monitored by virtue of the decrease in absorption of NADPH at 340 nm. 5.3.1. Determination of the optimum pH of activity of the purified yeast recombinant isomeroreductase
• the optimum pH of activity of this enzyme was determined, by measuring the activity of the purified enzyme in buffers of variable pH.
• the optimum activity pH for yeast isomeroreductase is 7.5, while studies have shown that for plant isomeroreductase is 8.2.
• This difference in optimum pH of isomeroreductase activity between the plant and the yeast is explained by the cellular localization of these 2 enzymes.
• the plant isomeroreductase is located in the chloroplast whose pH is 8.2 in the light, while the yeast isomeroreductase is located in the mitochondrion whose pH is 7.5.
• the optimum pH of activity of these isomeroreductases is therefore well suited to the cellular environment in which they are found.
• the specific activity of the yeast isomeroreductase for the AHB and AL substrates is 6 and 1 ⁇ mol of oxidized NADPH. Min “. Mg " of protein, respectively.
• the specific activities of the plant isomeroreductas for the AHB and AL substrates are also 6 and 1 ⁇ mol of oxidized ⁇ ADPH.min " .mg " of protein, respectively.
• Affinities for the cof actors ⁇ ADPH and ⁇ ADH The affinity of the yeast isomeroreductase for ⁇ ADPH is very high (FIG. 3).
• K M K M - 5 ⁇ M
• K M ⁇ ADPH K ⁇ ADPH obtained for the purified yeast isomeroreductase is consistent with the value obtained for the partially purified yeast enzyme (K M ⁇ 2.5 ⁇ M; Hawkes et al, 1989).
• K M ⁇ ADH
• ⁇ ADH 645 ⁇ M in the presence of AHB and Mg 2+ ; Dumas et al., Biochem. J., 288: 865-874, 1992), the yeast enzyme seems to be unable to use ⁇ ADH. No enzymatic activity was detected in the presence of ⁇ ADH (at 300 ⁇ M) under saturated conditions with AHB substrate and magnesium, perhaps because of an affinity for ⁇ ADH even more weak than that of plant isomeroreductase. The yeast isomeroreductase would use the
• NADPH as a donor of hydrogen with an even greater specificity than the plant enzyme.
• the study of the binding stoichiometry of the inhibitors N-hydroxy-N-isopropyloxamate and dimethylphosphinoyl-2-hydroxyacetate on the yeast isomeroreductase has shown that these inhibitors behave like irreversible inhibitors.
• Equation (1) therefore makes it possible to describe the kinetics of formation of the reaction product, this equation being applicable to irreversible inhibitors.
• the parameters ml, m2 and m3, defined from equation (1), are obtained directly from the adjustment of the experimental curves by the KaleidaGraph software, as well as the errors associated with the determination of these parameters. Equation (1):
• DO 3 0 is the optical density measured with a spectrophotometer at 340 nm at time "t"
• the apparent rate of formation of the enzyme-inhibitor complex is a linear function of the inhibitor concentration.
• the graphical representation m 3 as a function of the inhibitor concentration is a hyperbola.
• the graphical representation m 3 as a function of the inhibitor concentration is linear, suggesting that the inhibition of the yeast isomeroreductase by these products is done in a single step (FIG.
• k 0 s - rn 3 apparent rate of formation of the enzyme-inhibitor complex
• k o constant of association of the inhibitor with the enzyme
• k -0 constant of dissociation of the inhibitor with the enzyme
• the k -0 is then calculated using the equation (3) thanks to the KaleidaGraph program. Equation (3):
• This equation can be used for the inhibitor N-hydroxy-N-isopropyloxamate, but not for dimethylphosphinoyl-2-hydroxyacetate.
• the graphic representation l / m 3 as a function of the concentration of substrate is linear, but does not pass through the origin.
• the graphic representation l / m 3 as a function of the substrate concentration is linear and the value of the ordinate at the origin is negligible.
• the dimethylphosphinoyl-2-hydroxyacetate inhibitor may not be completely irreversible.
• a linear regression line then makes it possible to obtain the value of k 0 for dimethylphosphinoyl-2 -hydroxyacetate.
• the k 0 values corresponding to the inhibitors N-hydroxy-N-isopropyloxamate and dimethylphosphinoyl-2-hydroxyacetate are 12,433 M " .s " and 7721 M “ .s " , respectively.
• k 0 for N-hydroxy-N-isopropyloxamate 1,900 M " .s " and k 0 for dimethylphosphinoyl-
• the quaternary structure of the yeast isomeroreductase has been studied using two different approaches: mass spectrometry and gel filtration.
• yeast isomeroreductase 194- monomer of the yeast isomeroreductase, represented by the states of charge [M + 12H] to [M + 14H] is demonstrated on this same mass spectrum.
• the yeast isomeroreductase could therefore be in equilibrium between a monomeric form and a dimeric form.

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