Patent Application
20050260727
The present invention relates to crystals of a protoporphyrinogen IX oxidase, to processes for preparing crystals of a protoporphyrinogen IX oxidase, to processes for determining the structure of protoporphyrinogen IX oxidases alone or complexed with a ligand, and to processes for identifying ligands of the mitochondrial protoporphyrinogen IX oxidase, which may be used as herbicides. The present invention also relates to the use of the crystal structure described here of the Nicotiana tabacum mitochondrial protoporphyrinogen IX oxidase for generating protein models of related enzymes and to the identification of inhibitors of related proteins.
The enzymic activity of protoporphyrinogen IX oxidase (also referred to as “PPO” hereinbelow, for short) consists of catalyzing the oxidation Of protoporphyrinogen IX (formula I) to protoporphyrin IX (formula II) by molecular oxygen:
The protein is a representative of a highly conserved enzyme family for which membrane association is presumably achieved by way of a conserved, hydrophobic region in the protein (Arnould et al., 1999). In plants, there are two isoforms of the enzyme, namely the plastidic and the mitochondrial protoporphyrinogen IX oxidase (Lermontova et al., 1997). While the plastidic isoform is located in thylakoid membranes and the inner and outer plasma membranes, the mitochondrial form is located in the inner membrane of mitochondria.
Protoporphyrinogen IX oxidase is known to be an important target of peroxidizing herbicides, such as, for example, acifluorfen. Inhibition of the enzyme results in an accumulation of protoporphyrin (which forms by spontaneous reaction of protoporphyrinogen Ix. The decoupling of protoporphyrin formation and subsequent introduction of an iron ion, catalyzed by the protein ferrochelatase results in a loss of feedback control of haem biosynthesis by the final product haem. The photosensitive protophorphyrin IX accumulated thereby generates singulet oxygen which leads to cell death by way of peroxidation of lipids. The observed bleaching of photosynthetically active plant parts can be attributed to this effect (Lermontova et al., 1997). Protoporphyrinogen IX oxidase is therefore an enzyme particularly suitable for the search for further, improved herbicidal active compounds. In this context, in particular, the spatial structure of this protein is also of particular interest, which structure could make it possible to identify inhibitors of the enzyme and thus potential herbicides without complicated biochemical processes.
SEQ ID NO: 1: SEQ ID NO: 1 (PPO) depicts the Nicotiana tabacum PPO amino acid sequence with mutations 1M and L224Q:
It was the object of the present invention to provide the spatial structure of the crystalline form of mitochondrial protoporphyrinogen IX oxidase and, in particular, the spatial structure of the PPO ligand-binding pocket, in order to make it possible for the said structure to be used in the search for novel herbicidal active compounds.
The object was achieved by providing a crystal of the PPO complexed with the inhibitor 4-bromo-3-(5′-carboxy-4′-chloro-2′-fluorophenyl)-1-methyl-5-trifluoromethylpyrazole, the structural coordinates of this crystal and processes for identifying ligands of the PPO or inhibitors of the enzymic activity of this enzyme.
The present invention thus relates to the crystal of a PPO, in particular a plant PPO. The crystalline PPO according to the invention is preferably that from Nicotiana tabacum. Particular preference is given to the PPO according to the invention comprising the amino acid sequence according to SEQ ID NO: 1.
The crystal of a PPO according to the invention is preferably a PPO with the bound inhibitor 4-bromo-3-(5′-carboxy-4′-chloro-2′-fluorophenyl)-1-methyl-5-trifluoromethylpyrazole. The crystal according to the invention of a PPO with the bound inhibitor 4-bromo-3-(5′-carboxy-4′-chloro-2′-fluorophenyl)-1-methyl-5-trifluoromethylpyrazole has preferably the structural coordinates defined in Table 2.
The invention relates in particular to a crystal having the space group C222, and the cell constants a=119.1 Å, b=147.3 Å, c=127.0 Å (see also Table 1). Such a structure also comprises a crystal having the structural coordinates according to Table 2.
The invention also relates to a process for preparing a crystal according to the invention, in which a selenomethionine-substituted protein is used. The invention further relates to a process for preparing a crystal according to the invention, in which a PPO is recombinantly produced in a bacterial host (e.g. E. coli) in the presence of selenomethionine in the medium, the selenomethionine-substituted protein is obtained from the bacterial cells and purified and crystals of this protein are prepared. The crystals may be prepared by any common method, for example in a hanging drop.
The invention therefore also relates to a process for preparing a crystal according to the invention, in which the crystal is generated by vapour diffusion in a sitting drop, the crystallization solution comprising 10% polyethylene glycol 1000, 0.1 M sodium citrate/HCl, pH 4.0 and 0.2 M sodium chloride.
The three-dimensional structure was resolved with the aid of protein crystals accessible to X-ray structural analysis by means of anomalous dispersion at a single wavelength and fully refined at 2.9 Å resolution. The present invention thus also relates to the three-dimensional structure of the complex of PPO and bound inhibitor 4-bromo-3-(5′-carboxy-4′-chloro-2′-fluorophenyl)-1-methyl-5-trifluoromethylpyrazole, determinable on the basis of these structural coordinates.
The present invention therefore relates in particular to a crystal of a PPO as described above, which comprises a ligand-binding pocket which is defined by the following amino acids at a distance of 4.5 Å from the bound ligand: Arg98, Ala174, Gly175, Thr176, Cys177, Gly178, Leu334, Phe353, Gly354, Val355, Leu 356, Leu369 Gly370, Thr37, Leu372 Phe392, Phe439. The sequence and positional information refers to the amino acids according to SEQ ID NO: 1.
The present invention furthermore relates to the crystal of a PPO-ligand complex which comprises a ligand-binding pocket which is defined by the above-described amino acids and in which one or more of these amino acids have been mutated. These mutations are preferably conservative mutations in which a replacement with an amino acid with similar physical properties takes place.
The coordinates of Table 2 represent the coordinates of the atoms in Angstrom. The coordinates are a relative set of data of positions which define a structure in three dimensions. Thus it is possible for a completely different set of data of coordinates, which is based on a different origin and/or axes, to represent nevertheless an equal or identical structure. If the relative positions of the atoms of the structure are changed in a way that, with superposition with the corresponding coordinates according to Table 2, the square root of the mean square deviation of the atoms of the peptide backbone of the protein is less than 1.5 Å, preferably less than 1.0 Å, particularly preferably less than 0.5 Å, then usually a structure is likewise obtained which corresponds essentially to a structure according to Table 2, with respect to its structural characteristics and to its usability for structure-based identification of ligands of the protein. It should also be noted that changing the number and/or positions of the water molecules and/or substrate molecules of Table 2 does not fundamentally impair the usability of the structural coordinates according to Table 2.
It is therefore considered as being encompassed by the scope of the present invention, if the structural coordinates shown in Table 2 are shifted towards a different origin and/or different axes, or if the relative positions of the atoms of the structure are changed in a way that the square root of the mean square deviation of the atoms of the peptide backbone of the protein is, in comparison with the coordinates according to Table 2, less than 1.5 Å, preferably less than 1.0 Å, particularly preferably less than 0.5 Å, or if the number and/or positions of the water molecules and/or the substrate molecules are changed.
The structure of the PPO complex may be changed by binding of a ligand or inhibitor to the PPO complex. These changes are often restricted to side-chain conformations, but it is also possible for entire groups of amino acids to move with their peptide backbone. In addition, typical conformational changes occur in enzymes with unoccupied ligand-binding pocket or in enzymes whose ligand-binding pocket is occupied by inhibitors. Such changes are likewise encompassed by the present invention.
The skilled worker knows that various of the processes according to the invention described herein often need only parts of rather than the whole set of data according to Table 2. For example, it is possible, as will be described hereinbelow, to use selected coordinates of the PPO crystal for modelling a chemical compound based on the structural data of a crystal according to the invention. Frequently, only 5, preferably 10, particularly preferably 50 and very particularly preferably 100, atoms of the PPO structure suffice in order to model a chemical compound capable of interacting with the PPO. Accordingly, it is also possible for the other processes described herein such as, for example, the homology-based modelling and the homology-based structure elucidation, to use all or only parts of the structural coordinates according to Table 2.
The above-described aspects of the invention represent, individually or combined with one another, preferred embodiments of the invention.
In addition to the three-dimensional structure described herein of a crystal of a PPO, which structure is already immediately highly important for the search for ligands and thus also for inhibitors of the enzyme, the present invention also relates to the use of the said structure in determining the structures of proteins homologous to the present PPO. Thus, structural prediction currently works satisfactorily, i.e. without any extraordinary complication, if the structure of a homologous protein, i.e. a protein of the same origin, is known. Then it is possible to assume that a similar 3D structure is present and to use the known structure as a template in order to generate a model for the 3D structure of the other protein by means of homology-based modelling (“homology modelling”). Thus, the three-dimensional PPO structure according to the invention also makes it possible to predict the three-dimensional structure of a PPO from organisms other than Nicotiana tabacum by modelling. Such protein models may be used in the same way as the three-dimensional structure resolved here. It is then also possible, by comparing the differences in the amino acid sequences, to predict differences in the ligand-binding pockets of the PPOs of various organisms. This is useful if specific ligands for specific organisms are searched for or if, in the reverse case, precisely unspecific ligands with broad action are searched for. In addition, the three-dimensional structure of the invention may also serve to generate protein models of other sequence-related enzymes.
If thus X-ray structural data for a PPO, for example from a different organism, for a complex of a PPO and a ligand are available, the structural data according to Table 2, provided here, may be used in order to interpret the obtained data in a simple manner and to produce a structure. For this purpose, processes are used which are familiar to the skilled worker. One process which may be used for this purpose is referred to as molecular replacement. Owing to this method, the new structure may be determined more rapidly and efficiently than when trying to produce this structure ab initio. Examples of suitable computer programs which are commonly used, are CNX (Brunger et al., 1998) and AMORE (Navaza, 1994).
In this context, the degree of homology of two protein structures correlates well with the degree of sequence identity. Usually, the method of molecular replacement may be used already with 40% identity between the sequence of the template and the sequence of the structure to be resolved. The term “homology” or “identity” is to be understood as meaning the number of amino acids corresponding to other proteins (identity), expressed in percentage. The identity is preferably determined by comparing a given sequence to other proteins with the aid of computer programs. If sequences which are compared to one another have different lengths, the identity is to be determined so as for the number of amino acids shared between the shorter sequence and the longer sequence to determine the percentage identity. The identity may be determined in a standard manner by means of known and publicly available computer programs such as, for example, ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW, for example, is made publicly available by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyer-hofstrasse 1, D 69117 Heidelberg, Germany. ClustalW may likewise be downloaded from various Internet sites, inter alia at IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, B. P.163, 67404 Ilikirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and at EBI (ftp://ftp.ebi.ac.ukipub/software/) and at all mirrored Internet sites of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB 10 1SD, UK). If the ClustalW computer program, Version 1.8, is used, in order to determine the identity between, for example, a given reference protein and other proteins, the following parameters are to be set: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP. One possibility of finding similar sequences is carrying out sequence database searches. Here, one or more sequences are provided in a “query”. This query sequence is then compared to sequences contained in the selected databases by means of statistical computer programs. Such database queries (“blast searches”) are known to the skilled worker and may be carried out with various providers. If such a database query is carried out, for example, with the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/), the standard settings given for the particular comparative query should be used. For protein sequence comparisons (“blastp”), these are the following settings: Limit entrez=not activated; Filter=low complexity activated; Expect value=10; word size=3; Matrix=BLOSUM62; Gap costs: Existence=11, Extension=1. The result of such a query lists, besides other parameters, also the proportion of identity between the query sequence and the similar sequences found in the databases. A protein according to the invention is therefore, in connection with the present invention, to be understood as meaning those proteins which, when at least one of the above-described methods for determining identity is used, have an identity of at least 40%, preferably of at least 50%, particularly preferably of at least 60%, further preferably of at least 70%, and in particular of at least 80%.
The present invention therefore also relates to a process for determining the structure of a protein having the enzymic activity of a PPO, which has preferably an identity of at least 40%, preferably of 50%, particularly preferably of 60%, and very particularly preferably of 70%, to the sequence according to SEQ ID NO: 1, which process comprises the following steps:
The term “homologous region(s)” refers to amino acid residues of two sequences, which are identical or similar (e.g. aliphatic, aromatic, polar, negatively charged, positively charged, etc.). Such residues are frequently referred to as “invariant” or “conserved”.
Preferably, any or all of steps (a) to (c) are carried out by means of computer-assisted modelling. This homology modelling is a process known to the skilled worker (see, for example, Greer, 1985 and Blundell et al., 1988).
The structures of amino acids located in non-conserved regions may be arranged manually by using the usual peptide geometry or simulation techniques, such as, for example, molecular dynamics computer simulation. The final step of the process is the improvement (refinement) of the overall structure by molecular dynamics and/or energy minimization.
The present invention also comprises mutants, with this term also referring to polypeptides which are obtained by replacing at least one amino acid of the PPO according to SEQ ID NO: 1 with another amino acid and/or by insertion and/or deletion of one amino acid and which still have the essentially same three-dimensional structure as the PPO according to Table 2. An essentially same structure here refers to a set of structure coordinates for which the square root of the mean square deviation is less than or equal to 2.0 Å when the structural coordinates are superimposed by the structural coordinates according to Table 2 and when this superposition contains at least 50%, preferably 70% and particularly preferably all of the Cα atoms of the PPO of the known structure.
Using the PPO crystal structure according to the invention enables databases which contain the structures of a large number of compounds which are interesting as ligands of a PPO or as inhibitors of the enzymic activity of a PPO (“candidate molecules”) to be screened with the aid of established automated computer processes (virtual screening). These candidate molecules are in general small organic (low-molecular-weight) molecules which are suitable for use as active compounds, for example in crop protection. Such active compounds should be accessible to chemical synthesis. It is possible to use, for example, algorithms such as FLEXX (Rarey et al., 1996) or GOLD (Jones et al., 1997) for virtual screening. This procedure enables compounds to be identified whose three-dimensional structure enables them to get into the binding pocket and to bind there, for example by forming hydrogen bonds, by hydrophobic interaction, by charge interactions, by van-der-Waals interactions or by dipole interactions. The compounds identified in this way may be synthesized and then be used as herbicides, for example.
The present invention therefore also relates to a process for identifying ligands of the PPO or inhibitors of its enzymic activity, which process comprises the following steps:
Preference is given to using the data of Table 2 for producing the three-dimensional structure of a PPO. However, it is also possible to use in the same way the structural coordinates of other PPOs homologous to the present PPO according to SEQ ID NO: 1, which have been determined using a process as described above, based on the structural coordinates according to Table 2.
As an alternative to this, it is also possible to use only the coordinates of the atoms of the PPO according to Table 2, which are located in spatial proximity of the binding region of the putative ligand or inhibitor. These may be utilized in order to model a ligand-binding pocket in which the putative ligand or inhibitor can bind. These coordinates are thus used for defining a space which may then be utilized “in silico” for screening candidate molecules with the aid of suitable computer-based processes, as indicated above.
The present invention therefore also relates to a process for identifying ligands of the PPO or inhibitors of its enzymic activity, which process comprises the following steps:
Preference is given to using the data of Table 2 for producing the selected coordinates of a PPO. However, it is also possible to use in the same way the coordinates of other PPOs homologous to the present PPO according to SEQ ID NO: 1, which have been determined by a process as described above, based on the structural coordinates according to Table 2.
Usually, it will be advantageous if the model of the ligand-binding pocket is based on a sufficient number of atoms of a PPO. Preference is therefore given to using the coordinates of atoms of the residues Arg98, Phe392, Leu356 and Leu372. Particular preference is given to using the coordinates of atoms of the residues Arg98, Ala174, Gly175, Thr176, Cys177, Gly178, Leu334, Phe353, Gly354, Val355, Leu 6, Leu369, Gly370, Thr371, Leu372, Phe392, Phe439 according to SEQ ID NO: 1.
The term “fitting” describes the computer-assisted or partially computer-assisted determination of the interaction between at least one atom of the candidate molecule and at least one atom of the PPO and of calculating the stability of this interaction. Various processes known to the skilled worker are available for this fitting.
Another application of the three-dimensional structure of the PPO complex according to the invention is the generation of new ligands. To this end, structural formulae for new ligands which can enter the binding pocket and bind there, for example by forming hydrogen bonds, by hydrophobic interaction, by charge interactions, by van-der-Waals interactions or by dipole interactions, are generated using the structure and with the aid of established de novo design programs on a computer. Examples of possible de novo design programs are LUDI (Böhm, 1992), LEGEND (Nishibata & Itai, 1991) and GROW (Moon & Howe, 1991). Compounds generated in such a way may be synthesized and then likewise be used as herbicides, for example.
Ligands or potential inhibitors which either have been found in an in silico screening process as described above or have been generated and synthesized de novo as described above may be checked for their herbicidal activity in a subsequent step.
The processes according to the invention therefore comprise, where appropriate, also the following further steps:
Alternatively, the ligands or potential inhibitors may also be contacted directly, where appropriate in a suitable formulation, with a plant or plant cells and the growth-inhibiting or killing action of the compounds on this plant may be determined.
Instead of or in addition to the said in vitro or in vivo processes for checking the identified compounds, the abovementioned processes may also comprise the following further steps:
In this way it is possible to obtain detailed information on binding of the ligand and potential inhibitor. This information may also be utilized in order to adjust the structure of the ligand, for example in order to improve its effectiveness and/or specificity, for example for the PPO of a particular organism. The above-described steps of selecting a ligand by screening or its de novo generation, of testing in a biochemical in vitro or in an in vivo assay, and the analysis of the complex by means of determining the structure may also be repeated several times in order to gradually improve the characteristics of the ligand or inhibitor.
In this context, it is possible to determine the structure of the complex of a PPO and an identified ligand or inhibitor by means of the abovementioned process based on the structural coordinates according to Table 2 for determining the structure of a homologous PPO or of a new complex of a PPO according to SEQ ID NO:1 and a new ligand in an efficient manner. This may involve introducing a new ligand into PPO crystals already present by “soaking” in a manner known to the skilled worker. However, PPO and ligand may also be co-crystallized.
Depending on their particular physical and/or chemical properties, the identified active compounds may be transferred to the usual formulations such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols and microencapsulations in polymeric substances and in coating compositions for seeds, and ULV cool and warm fogging formulations.
These formulations are produced in a known manner, for example by mixing the active compounds with extenders, that is liquid solvents, liquefied gases under pressure, and/or solid carriers, optionally with the use of surfactants, that is emulsifiers and/or dispersants, and/or foam formers. If the extender used is water, it is also possible to employ, for example, organic solvents as auxiliary solvents. Essentially, suitable liquid solvents are: aromatics such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, alcohols such as butanol or glycol and their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents such as dimethylformamide or dimethyl sulphoxide, or else water. Liquefied gaseous extenders or carriers are to be understood as meaning liquids which are gaseous at standard temperature and under atmospheric pressure, for example aerosol propellants such as halogenated hydrocarbons, or else butane, propane, nitrogen and carbon dioxide. Suitable solid carriers are: for example ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals such as finely divided silica, alumina and silicates. Suitable solid carriers for granules are: for example crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite, or else synthetic granules of inorganic and organic meals, and granules of organic material such as sawdust, coconut shells, maize cobs and tobacco stalks. Suitable emulsifiers and/or foam formers are: for example nonionogenic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates, or else protein hydrolysates. Suitable dispersants are: for example lignosulphite waste liquors and methylcellulose.
Tackifiers such as carboxymethylcellulose, natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, or else natural phospholipids such as cephalins and lecithins and synthetic phospholipids can be used in the formulations. Other possible additives are mineral and vegetable oils.
It is possible to use colorants such as inorganic pigments, for example iron oxide, titanium oxide and Prussian Blue, and organic dyestuffs such as alizarin dyestuffs, azo dyestuffs and metal phthalocyanine dyestuffs, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.
The formulations generally comprise between 0.1 and 95 percent by weight of active compound, preferably between 0.5 and 90%.
The active compounds according to the invention can, as such or in their formulations, also be used in a mixture with known fungicides, bactericides, acaricides, nematicides or insecticides, to broaden, for example, the activity spectrum or to prevent development of resistance. In many cases, synergistic effects are obtained, i.e. the activity of the mixture is greater than the activity of the individual components.
When using the compounds according to the invention as fungicides, the application rates can be varied within a relatively wide range depending on the application.
According to the invention, it is possible to treat all plants and parts of plants. Plants are to be understood here as meaning all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods, including the transgenic plants and including plant cultivars which can or cannot be protected by plant breeders' certificates. Parts of plants are to be understood as meaning all above-ground and below-ground parts and organs of plants, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stems, trunks, flowers, fruit-bodies, fruits and seeds and also roots, tubers and rhizomes. Parts of plants also include harvested material and vegetative and generative propagation material, for example seedlings, tubers, rhizomes, cuttings and seeds.
The treatment of the plants and parts of plants according to the invention with the active compounds is carried out directly or by action on their environment, habitat or storage area according to customary treatment methods, for example by dipping, spraying, evaporating, atomizing, broadcasting, brushing-on and, in the case of propagation material, in particular in the case of seeds, furthermore by one- or multilayer coating.
The following examples illustrate various aspects of the present invention and are not to be construed as limiting.
Protein Preparation and Crystallization
During proteolytic cleavage of the fusion protein used, a further cleavage within the PPO was observed, in addition to the desired removal of the N-terminal part. In order to suppress this undesired secondary reaction, lysine 224 was mutated to glutamine 224. Nicotiana tabacum PPO was expressed in the form of fusion proteins with thioredoxin and a hexahistidine part with the aid of the expression vector pET32a. The bacterial strain used for this purpose was B834(DE3). The cells were cultured in minimal medium which contained selenomethionine instead of methionine. After the cells had been disrupted, a three-stage purification protocol was used to isolate the PPO. For this purpose, the cell lysate was purified in a first step by column chromatography on an Ni-NTA gel. After the amino-terminal fusion part had been proteolytically removed, the protein was subsequently chromatographically purified via a gel filtration column (Sephacryl S-200) and then via an anion exchange column (ResourceQ). For crystallization, the protein was transferred to a buffer containing 5 mM Tris/HCl, pH 8.0, 0.1% Triton X-100 and 50 mM sodium chloride. The crystals were generated by the method of vapour diffusion in a hanging drop. The crystallization solution used contained 10% polyethylene glycol 1000, 0.1 M sodium citrate/HCl, pH 4.0 and 0.2 M sodium chloride. Crystals with dimensions of 150×100×100 μm3 formed within 2 days. The intensely yellow crystals belonged to the C2221 space group and had axis lengths of a=119.1 Å, b=147.3 Å and c=127.0 Å.
Crystallographic Data Recording and Analysis
The crystals which initially diffracted only to a resolution of approx. 8 Å were transformed into a different crystal form with the aid of a Free-Mounting System (Proteros biostructures GmbH, Martinsried, Germany). X-ray diffraction data were recorded at a wavelength of 0.9790 Å at the ESRF (European Radiotion Synchrotron Facility) in Grenoble, France. The diffraction images were processed using the program XDS (Kabsch, 1993). Anomalous scattering centres were identified by the program SnB (Weeks & Miller, 1999). Heavy metal atom parameters were refined using the program SHARP (Fortell & Bricogne, 1997). The structural model was modelled to the resulting electron density using the program o (Jones et al., 1991). Iterative refinement of the model was carried out using the program CNS (Brünger et al., 1998) (cf. Table 1).
Architecture of the Inhibitor Complex of the PPO
The 503 amino acids of the PPO form a compact overall structure which comprises three domains: an FAD-binding domain, a substrate-binding domain with a topology similar to p-hydroxybenzoate hydrolase and a membrane-binding domain. The structural moieties involved in FAD binding exhibit substantial sequential and structural homologies to other flavoenzymes such as human monoamine oxidase B (Binda et al., 2002), with an rmsd (root mean square deviation) of 1.68 Å over 256 Ca atoms and a sequence identity of 15.1%, polyamine oxidase (Binda et al., 1999), with an rmsd of 1.71 Å over 190 Ca atoms and a sequence identity of 14.9% and D-amino acid oxidase (Mitsutani et al., 1996), with an rmsd of 1.98 Å over 148 Ca atoms and a sequence identity of 14.9%. The model implies that the eight helices, α4 to α11, which include the conserved sequence region from residue 150 to residue 213 represent the membrane anchor and are presumably monotopically embedded in the membrane. In this model, predominantly apolar residues are oriented towards the hydrophobic moiety of the membrane. Charged residues which are likewise located in the membrane-embedded surface region are involved in the formation of salt bridges or may reach the positively charged sugar phosphate region of the membrane. These residues are exclusively lysines or histidines. In the PPO crystal structure, the membrane-binding domains of in each case four protomers are in contact with one another, with two contacts covering in each case 800 Å3 of solvent-accessible surface. Two further contacts each covering 400 Å3 of surface exist perpendicularly thereto. The latter are presumably the contacts which build the biologically relevant dimer. This interaction could be stabilized in vivo additionally by phospholipids of the surrounding membrane. In the dimeric arrangement of two PPO protomers, the active sites are on the outside of the dimer, close to the membrane surface. The dimer contact area is formed by the helices α5 and α14, with substantial contributions from leucine residues which interact with the corresponding leucine residues in the second monomer. This results in a T-shaped dimer in which two membrane-binding domains form a hydrophobic surface area which is extraordinarily suitable for insertion into the membrane. This architecture is similar to the likewise monotopically membrane-bound prostaglandin H2 synthase (Loll et al., 1995).
The Active Site and the Structural Basis of Catalysis
The active site of the PPO is located between the FAD- and the substrate-binding domain. The inhibitor binds to the active site in a tunnel-shaped inversion close to the FAD cofactor in a stretched conformation (
In Vitro Assay for Determining the Enzymic Activity of a PPO in the Presence and Absence of a Ligand
The enzymic activity of the PPO was checked by firstly preparing a solution of freshly reduced protoporphyrinogen IX. For this purpose, 1.5 mg of commercially available protoporphyrin IX were admixed with 8 ml of 10 mM KOH and 2 ml of absolute ethanol. This solution was degassed using argon and added to 1.5 g of 5% strength sodium amalgam weighed under protective gas. The mixture was left at room temperature for 15 minutes and then titrated with 40% strength phosphoric acid to a pH of 7.0. 5 ml of the resulting solution were added through a sterile filter (pore size 0.45 μm, diameter 1 cm) to 25 ml of buffer solution containing 100 mM K2HPO4, 100 mM KH2PO4. 1 mM EDTA and 0.3% (v/v) TWEEN 80 at a pH of 7.2. The enzymic reaction was started by adding in each case 50 μl of enzymic solution (10 μg/ml in 5 mM Tris/HCl, 50 mM NaCl, pH 8.0) to 250 μl of the resulting reaction solution. The reaction progress was monitored by observing the fluorescence after 0, 5, 15 and 30 minutes, using a fluorescence spectrometer at an excitation wavelength of 410 nm and an emission wavelength of 635 nm. The effect of unspecific oxidation of the substrate by atmospheric oxygen was corrected by measuring a sample which did not contain any protein in parallel in the same manner. The activity resulting from this measurement was subtracted from the total activity in order to obtain the specific activity.
In Vivo Assay for Determining the Herbicidal Activity of a Ligand (Post-Emergence Assay)
To produce a preparation of active compound, containing a candidate compound, 1 part by weight of active compound is mixed with the stated amount of solvent, the stated amount of emulsifier is added and the concentrate is diluted with water to the desired concentration.
Test plants which are from 5 to 15 cm in height are sprayed with the preparation of active compound in such a way that the amounts of active compound per unit area, desired in each case, are applied. The concentration of the spray liquor is chosen in such a way that the amounts of active compound desired in each case are applied in 1000 l of water per ha.
After three weeks, the degree of damage to the plants is rated in % damage in comparison to the development of the untreated control. The figures then denote: 0%=no effect (as untreated control), 100%=total destruction.
Values in parentheses correspond to the last shell of resolution
*Rsym (I) = Σhkl Σi | Ihkl,i − < Ihkl > | / Σhkl Σi | Ihkl,i | with <Ihkl > mean intensity of multiple Ihkl,i observations of symmetry-equivalent reflections.
Mean square deviations based on ideal values.
§Rcryst = Σhkl | Fobs − Fcalc | / Σhkl |Fobs with Fobs and Fcalc observed and calculated structural amplitudes, respectively. Rfree is Rcryst, but calculated with those 10% of data not considered in the refinement.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102004012730.1 | Mar 2004 | DE | national |
| 102004025412.5 | May 2004 | DE | national |
