Methods for identifying herbicidal compounds

Abstract

The present invention relates to an in vitro method for determining whether a candidate compound is an inhibitor of diaminopelargonic acid aminotransferase (DAPA-AT), comprising: (i) assaying the candidate compound in an assay mixture comprising (a) a DAPA-AT, (b) an amine donor, (c) pyridoxal 5'-phosphate (PLP), and (d) 7-keto-8-aminopelargonic acid (KAPA); and (ii) detecting inhibition of DAPA-AT by the candidate compound, wherein the amine donor comprises an aliphatic diamine or aliphatic polyamine.

Description

[Technical Field]

[0001] Biotin, also known as vitamin B7, is an essential cofactor for enzymes involved in cellular processes, including the metabolism of fats, proteins, and carbohydrates. Plants and most fungi / bacteria can synthesize biotin, in contrast to animals, which obtain it from food sources or intestinal bacteria. Therefore, inhibiting enzymes in the biotin synthesis pathway in plants is an attractive herbicide target. [Background technology]

[0002] In bacteria, biotin is synthesized from pimeloyl-CoA and alanine through the activity of four enzymes (BioF, BioA, BioD, and BioB) encoded by genes located within an operon. BioF, also known as 7-keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), catalyzes the production of KAPA from pimeloyl-CoA and L-alanine. BioA, also known as 7,8-diaminopelargonic acid aminotransferase (DAPA-AT) or DAPA synthase (EC 2.6.1.62), then carries out the next step, converting KAPA to 7,8-diaminopelargonic acid (DAPA). BioD, also known as dethiobiotin synthetase (DTBS) (EC 6.3.3.3), subsequently converts DAPA to dethiobiotin (DTB), which is then converted to biotin via the activity of BioB, also known as biotin synthase (EC 2.8.1.6).

[0003] In plants, DAPA-AT corresponds to BIO1, the orthologue of BioA, and DTBS corresponds to BIO3, the orthologue of BioD. The genes encoding BIO1 and BIO3 reside at a single locus within the plant genome, and essentially, both single transcripts and chimeric BIO3-BIO1 transcripts can arise through alternative splicing events to produce either bicistronic transcripts capable of generating individual BIO3 and BIO1 proteins; or monocistronic transcripts producing the bifunctional fusion protein BIO3-BIO1, also known as BioDA (dethiobiotin synthetase / 7,8-diaminopelargonate aminotransferase). However, the BIO3-BIO1 fusion protein is thought to be the major, if not the only, protein produced by the BIO3-BIO1 locus in plants. Thus, BIO3-BIO1 is a single bifunctional enzyme that catalyzes two independent steps in biotin biosynthesis. Inhibition of the BIO3-BIO1 enzyme, and in particular the BIO1 catalytic domain, therefore represents an attractive herbicide target.The present invention therefore relates to methods for identifying inhibitors of DAPA-AT.

[0004] DAPA-AT is a PLP (pyridoxal 5'-phosphate)-dependent enzyme, and several biochemical assays for DAPA-AT activity have been developed and reported. While many of these assays have been developed using the bacterial BioA enzyme, Cobessi et al. (2012) The Plant Cell, Vol. 24:1608-1625 described the development of an assay using the Arabidopsis BIO3-BIO1 enzyme. However, a common feature of all of these assays reported to date is the use of S-adenosyl-L-methionine (SAM) as the amino group donor, which is converted to S-denosyl-4-methylthio-2-oxobutanoate (Scheme 1). Scheme 1: Activity of DAPA-AT (DAPA synthase) with SAM as the amine donor. [ka] Summary of the Invention [Means for solving the problem]

[0005] The present invention is based on the development of a new assay in which alternative amine donors are utilized.

[0006] Thus, the present invention provides an in vitro method for determining whether a candidate compound is an inhibitor of diaminopelargonic acid aminotransferase (DAPA-AT), comprising: (i) assaying the candidate compound in an assay mixture comprising (a) DAPA-AT, (b) an amine donor, (c) pyridoxal 5'-phosphate (PLP), and (d) 7-keto-8-aminopelargonic acid (KAPA); and (ii) detecting inhibition of DAPA-AT by the candidate compound, wherein the amine donor comprises an aliphatic diamine or aliphatic polyamine or a carbamate derivative thereof.

[0007] Aliphatic diamines refer to low molecular weight aliphatic compounds containing two amino (NH) groups, such as cadaverine or putrescine. Aliphatic polyamines refer to low molecular weight aliphatic compounds containing three or more amino (NH) groups, such as spermidine or spermine. Thus, in a preferred embodiment of the present invention, the amine donor is represented by the formula (I): [ka] Compounds of wherein X is NH or CH; R 1 is hydrogen or -[CH2]pNH2; m and n are independently selected from 0, 1, 2, 3, or 4; and p=1, 2, 3 or 4) or R 1is —C(O)OH;

[0008] In another embodiment of the present invention, the amine donor is cadaverine (X=CH, R 1 = H, m = 2, n = 2), putrescine (X = CH2, R 1 = H, m = 2, n = 1), spermidine (X = NH, R 1 = H, m = 3, n = 4) and spermine (X = NH, R 1 =-[CH2] p NH2, m=3, n=4, p=3). In a particularly preferred embodiment of the present invention, the amine donor is spermidine.

[0009] It has been discovered that the use of an aliphatic diamine or polyamine donor (rather than SAM), particularly spermidine, can provide a significantly improved assay, particularly for assays utilizing plant BIO1 or BIO3-BIO1 enzymes (see Figure 1). Without being bound by any particular theory, it is believed that when spermidine is utilized in the assay, it is converted to 4-(3-aminopropylamino)butanal (or 3-(4-aminobutylamino)propanal) (Scheme 2).

[0010] Scheme 2: Activity of DAPA-AT (DAPA synthase) with spermidine as the amine donor. [ka]

[0011] The provision of new practical assays would therefore be particularly useful in methods for identifying inhibitors of DAPA-AT, particularly plant BIO1 or BIO3-BIO1, which may aid in the discovery and development of herbicidal compounds.

[0012] The candidate compound can be any compound that is a putative inhibitor of DAPA-AT. Examples of known DAPA-AT inhibitors that can be used to develop this method include herbicidal cinnoline compounds, such as ethyl 5-acetyl-4-oxo-1-(2,2,3,3-tetrafluoro-1,4-benzodioxin-6-yl)cinnoline-3-carboxylate (Compound A); herbicidal compounds, such as [5-carboxy-1-(1-hydroxyethyl)pentyl]ammonium; chloride (compound B); herbicidal pyridone compounds, such as 2-(3,4-dichlorophenyl)-5-(2,4-difluorophenyl)-1-ethyl-6-methyl-4-oxo-pyridine-3-carboxylic acid (compound C) or 5-[2-chloro-5-(trifluoromethoxy)phenyl]-2-(3,4-dichlorophenyl)-1-ethyl-6-methyl-4-oxo-pyridine-3-carboxylic acid (compound D); herbicidal compounds, such as [(1R)-2-(6-carboxypyridin-1-ium-2-yl)oxy-1-methyl-ethyl]ammonium; dichloride (compound E); herbicidal pyrrolidin-2-ones, such as 2-[1-[(2,3-difluorophenyl)methyl]-5-oxo-pyrrolidin-2-yl]-N-(2-methyl-1,2,4-triazol-3-yl)acetamide (compound F), 2-[1-[(2,3-difluorophenyl)methyl]-5-oxo-pyrrolidin-2-yl]acetic acid (compound G), 2-[5-oxo-1-[(2,3,5-trifluorophenyl)methyl]pyrrolidin-2-yl]acetic acid (compound H), 2-(4-fluorophenoxy)ethyl 2-[1-[(2,3-difluorophenoxy)methyl]pyrrolidin-2-yl]acetic acid (compound H), N-benzylazole herbicidal compounds, such as 2-(3,4-dichlorophenyl)-1-ethyl-4-oxo-6-[[3-(trifluoromethyl)pyrazol-1-yl]methyl]pyridine-3-carboxylic acid (compound L);and herbicidal quinolone compounds, such as 2-(3,4-dichlorophenyl)-1-ethyl-6-fluoro-4-oxo-quinoline-3-carboxylic acid (Compound M). Results obtained using these compounds to develop the method of the present invention are shown in Table 1, and typical IC50 plots are shown in Figure 2.

[0013] The assay mixture contains (a) DAPA-AT, (b) an amine donor, (c) pyridoxal 5'-phosphate (PLP), and (d) 7-keto-8-aminopelargonic acid (KAPA). The term diaminopelargonic acid aminotransferase (DAPA-AT) represented by component (a) refers to an enzyme that catalyzes the conversion of 7-keto-8-aminopelargonic acid (KAPA) to 7,8-diaminopelargonic acid (DAPA). Examples of DAPA-ATs include, for example, bacterial BioA enzymes or functional derivatives or fragments thereof. More preferably, the DAPA-AT is a plant BIO1 enzyme, which can exist in the form of a bifunctional BIO3-BIO1 enzyme or a functional derivative or fragment of the BIO1 enzyme or the BIO3-BIO1 enzyme. BIO3-BIO1 refers to an enzyme that can catalyze the conversion of KAPA to dethiobiotin. The exact nature of the DAPA-AT is irrelevant to the present invention, and many suitable DAPA-AT enzyme sequences have been reported in the literature. Thus, the methods of the present invention can utilize a variety of plant BIO3-BIO1 enzymes, including, for example, mature BIO3-BIO1 enzymes from monocotyledonous plant species (e.g., Setaria italica—see FIG. 3 for results) and dicotyledonous plant species (e.g., Arabidopsis thaliana—see FIG. 1 for results).

[0014] Thus, in a preferred embodiment of the present invention, the DAPA-AT is a functional derivative of plant BIO1 or a fragment thereof. In a more preferred embodiment, the DAPA-AT is provided within a bifunctional BIO3-BIO1 enzyme or a functional derivative or fragment thereof. In a preferred embodiment, the DAPA-AT is a BIO3-BIO1 enzyme selected from the group consisting of SEQ ID NOs: 1-37, or a functional derivative or fragment thereof. In one embodiment of the present invention, the DAPA-AT is mature Arabidopsis BIO3-BIO1 (SEQ ID NO: 21) or Setaria BIO3-BIO1 (SEQ ID NO: 25), or a functional derivative or fragment thereof. In one embodiment of the present invention, the DAPA-AT is a plant BIO3-BIO1 enzyme that is not derived from Arabidopsis.

[0015] A functional derivative refers to a mutant in which some amino acids in the wild-type DAPA-AT have been substituted and / or deleted, but which retains DAPA-AT activity. Typically, such functional derivatives retain 85%, more preferably 90%, even more preferably 95%, and even more preferably 99% sequence identity at the amino acid level.

[0016] Fragment refers to a truncated form of the DAPA-AT enzyme that retains DAPA-AT activity. For example, it should be understood that the wild-type BIO3-BIO1 enzyme contains an N-terminal mitochondrial targeting peptide (e.g., 22 amino acids for the Arabidopsis BIO3-BIO1 of SEQ ID NO: 1), which can be removed such that a truncated mature DAPA-AT is used in the methods of the invention. Examples of BIO3-BIO1 enzymes from which the putative N-terminal mitochondrial targeting peptide has been removed are provided as SEQ ID NOs: 21-37.

[0017] It is envisioned that DAPA-AT can be provided for use in the assay in a number of art-recognized ways, for example, it can be provided in the form of a crude extract, but more preferably, the DAPA-AT will be produced by recombinant means and used in the assay in an appropriately purified form.

[0018] The amine donor can be obtained from any suitable source. For example, the amine donor can be obtained from a suitable biological source and contained, for example, in a crude extract or clarified biological lysate. Alternatively, preferably, the amine donor can be chemically synthesized and thus provided in the assay mixture in an appropriately pure form. It should be understood that component (b) can contain two or more amine donors.

[0019] Because DAPA-AT is a PLP-dependent enzyme, it is necessary to further include (c) PLP in the assay mixture. PLP may already be present bound to DAPA-AT and thus may be provided in the assay mixture in this manner. Alternatively, exogenous PLP may be provided in the assay mixture, which is readily available from commercial sources.

[0020] The substrate 7-keto-8-aminopelargonic acid (KAPA) (d) is also readily available from commercial sources.

[0021] Those skilled in the art will appreciate that additional assay components may be included in the assay mixture, including, for example, buffers such as 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]glycine (Tricine) or 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), salts such as sodium chloride, potassium chloride or magnesium chloride, detergents such as polyoxyethylene(20) sorbitan monolaurate (Polysorbate 20), 2- Other examples of suitable ion exchange media include [4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100) or 3-{dimethyl[3-(3α,7α,12α-trihydroxy-5β-cholan-24-amido)propyl]azaniumyl}propane-1-sulfonate (CHAPS), reducing agents such as (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol (dithiothreitol), chelating agents such as N,N'-(ethane-1,2-diyl)bis[N-(carboxymethyl)glycine] (EDTA), or other proteins such as bovine serum albumin (BSA). The exact nature of these general components is not relevant to the present invention.

[0022] Advantageously, the method of the present invention will be carried out in at least two separate steps. In the first step, a candidate compound is introduced into an enzyme mixture containing (a) DAPA-AT. The enzyme mixture is then optionally incubated for a period of time to allow the candidate compound to bind to the DAPA-AT. In the second step, an assay mixture is provided by adding a substrate mixture containing an amine donor (b) and KAPA (d) to the enzyme mixture. PLP (c) is usually included in the enzyme mixture, but can additionally / alternatively be provided in the substrate mixture. The assay will usually be carried out at room temperature for 60 minutes.

[0023] The "detecting inhibition" portion of the method can be carried out using several art-recognized methods that will be readily apparent to those skilled in the art. For example, options include direct detection of DAPA formation or KAPA reduction in these practical assays or by coupling the assay to dethiobiotin synthetase (DTBS) and detecting the formation of the product, DTB. In situations where BIO3-BIO1 is utilized in the assay, the assay mixture can further include (e) ATP and (f) CO2 to detect the formation of DAPA and / or DTB. These components can be monitored, for example, using an appropriate LC-MS procedure. Other assays, including radiolabeled and spectrophotometric assays, are also contemplated. However, in preferred embodiments of the present invention, DAPA formation is monitored in the assay. In more preferred embodiments, DAPA is conveniently converted to a fluorescent derivative, such as DAPA:o-phthalaldehyde. [ka]

[0024] The use of fluorescent derivatives in the methods of the invention is particularly useful because they are suitable for high-throughput assays, for example, using multi-well plates. Thus, in certain embodiments of the invention, the assay is stopped using a stop mixture comprising o-phthalaldehyde and 2-mercaptoethanol.

[0025] Inhibition can be determined by comparing the results obtained from an assay with the results obtained from an otherwise identical assay performed in the absence of the candidate compound. % inhibition can then be calculated to characterize whether a candidate compound is an inhibitor of DAPA-AT using the following calculation: % inhibition = 100 x ((((MAX - MIN) - (X - MIN)) / (MAX - MIN))), where MIN and MAX are the average values ​​of the minimum and maximum response controls, and X is the sample of interest.

[0026] The present invention further provides a compound of formula (I) [ka] Compounds of wherein X is NH or CH; R 1 is hydrogen or -[CH2] p NH2; m and n are independently selected from 0, 1, 2, 3, or 4; and p=1, 2, 3 or 4); or R 1 of an amine donor, including its carbamate derivative, wherein is —C(O)OH; It was provided for use in an in vitro diaminopelargonate aminotransferase (DAPA-AT) assay. [Example]

[0027] The following non-limiting examples provide specific methods representative of the present invention.

[0028] Example 1 - Expression of Arabidopsis thaliana BIO3-BIO1 (BioDA) in E. coli The pET-24 plasmid containing the DNA sequence encoding Arabidopsis thaliana-derived BIO3-BIO1, codon-optimized for expression in E. coli, with the predicted N-terminal mitochondrial transit peptide removed, containing an N-terminal 6xHis tag and thrombin cleavage site, and under the control of a T7 promoter (SEQ ID NO: 38), was synthesized by Twist Bioscience. The plasmid was expressed in E. coli BL21(DE3)bioA and selected with 50 μg / mL kanamycin and 25 μg / mL chloramphenicol. An overnight culture grown at 37°C was used to inoculate 8 x 500 mL AIM in shake flasks at a 1:100 ratio. The culture was grown at 37°C and 200 rpm for 3.5 hours, then overnight at 20°C. Cells were harvested by centrifugation at 10,000 g for 30 minutes. Cells were stored at -80°C until extraction. A 30 g cell pellet was thawed in 120 mL of IMAC buffer A (20 mM HEPES, pH 7.5, 25 mM imidazole, 500 mM sodium chloride, 0.15 mM pyridoxal phosphate, 0.5 mM TCEP, 1× complete Protease Inhibitor Tablet (Roche)). The resuspension was passed through a cell disrupter at 18,000 PSI and centrifuged at 50,000 g for 25 minutes at 4°C. His-tagged BIO3-BIO1 proteins were purified from the extract supernatant using a HisTrap FF column on an AKTA avant chromatography system (Cytiva) and eluted in IMAC buffer B (20 mM HEPES pH 7.5, 500 mM imidazole, 500 mM sodium chloride, 0.15 mM pyridoxal phosphate, 0.5 mM TCEP). SEC was then performed using a HiLoad 26 / 60 Superdex200 column in SEC buffer (25 mM HEPES pH 7.5, 150 mM sodium chloride, 0.15 mM pyridoxal phosphate, 0.5 mM TCEP). Protein concentration was measured and calculated using absorbance at 280 nm. The extract was diluted 4-fold, and 10 μL was analyzed by SDS-PAGE.

[0029] Example 2 - DAPA-AT Assay To measure DAPA-AT enzyme activity in the presence of herbicides (candidate compounds), 0.5 μL of herbicide was incubated with 20 μL of enzyme mix (100 mM EPPS pH 8.6, 0.1 mM pyridoxal phosphate, 0.1 mg / mL BSA, 25 nM BIO3-BIO1 enzyme) in wells of a 384-well black plate at 20°C for 10 min, followed by the addition of 20 μL of substrate mix (final concentrations: 100 mM EPPS pH 8.6, 0.1 mM pyridoxal phosphate, 0.1 mg / mL BSA, 10 μM spermidine, 1 μM KAPA, 100 mM NaHCO3) and further incubation at 20°C for 60 min. The reaction was stopped by adding 50 μL of derivatization mix consisting of 185 mM boric acid NaOH pH 9.4, 3 mM o-phthalaldehyde, 6 mM β-mercaptoethanol, 22.5% (v / v) ethanol, and 3% (v / v) methanol. The fluorescent product generated by the reaction of DAPA with o-phthalaldehyde and β-mercaptoethanol was measured at 470 nm after excitation at 410 nm. Percent inhibition was calculated using the formula: % Inhibition = 100 × ((((MAX - MIN) - (X - MIN)) / (MAX - MIN))), where MIN and MAX are the average values ​​of the minimum and maximum response controls, and X is the test sample. Compounds are typically serially diluted 3-fold and added directly to the assay plate at 10 concentrations, with a final concentration of 100 μM. The results are shown in Table 1 below. Percent inhibition values ​​represent the compound concentration resulting in 50% inhibition (IC 50 To calculate the formula Y=Bottom+(Top-Bottom) / (1+((X Hill Slope ) / (I C 50 Hill Slope ))) is used to fit the dose-response curve.

[0030] [Table 1]

[0031] Example 3 - Measurement of DAPA-AT and dethiobiotin synthetase enzyme activity in the presence of different amine donors To measure DAPA-AT and dethiobiotin synthetase enzyme activity in the presence of different amine donors, 10 μL of candidate amine donors were placed in wells of a 96-well plate (final concentration: 3.3 mM). 10 μL of substrate mix (final concentrations: 100 mM EPPS pH 8.6, 0.1 mM pyridoxal phosphate, 1 mM ATP, 5 mM MgCl2, 20 μM KAPA, 100 mM NaHCO3) was added to each well. The assay was initiated by the addition of 10 μL of BIO3-BIO1 enzyme (final concentration: 1 μM) and incubated at 20 °C for up to 90 min. The reaction was stopped by the addition of 100 μL of 1:1 acetonitrile / methanol, and dethiobiotin (DTB) was quantified by LC-MS using an Orbitrap mass spectrometer in positive ionization and full scan mode with a heated electrospray ionization source. Analytes were separated by reversed phase chromatography using a UPLC 2.1 mm x 50 mm C18 column. A 5 μL sample was injected onto the column equilibrated with 95% solution A (0.2% formic acid) and 5% solution B (acetonitrile) at a flow rate of 0.7 mL / min. DTB was eluted with a 0.7 mL gradient from 5 to 95% B and compared with a commercially available standard of DTB. The results are summarized in Figure 1. [Brief explanation of the drawings]

[0032] [Figure 1] Activity of Arabidopsis BIO3-BIO1 comparing various amine donors. [Figure 2] Regular IC50 plot. [Figure 3] Setaria BIO3-BIO1 enzyme activity at different amine donor (spermidine) concentrations.

[0033] array BIODA / BIO3-BIO1 protein sequence - full length (putative mitochondrial targeting sequence underlined) Arabidopsis thaliana BioDA protein (SEQ ID NO: 1) [ka] Zea mays BioDA protein sequence (SEQ ID NO: 2) [ka] Nannochloropsis gaditana BioDA protein (SEQ ID NO: 3) [ka] Taxus chinensis BioDA protein (SEQ ID NO: 4) [ka] Physcomitrium patens BioDA protein (SEQ ID NO: 5) [ka] Adiantum nelumboides BioDA protein (SEQ ID NO: 6) [ka] Foxtail millet (Setaria italica) BioDA protein (SEQ ID NO: 7) [ka] Date palm (Phoenix dactylifera) BioDA protein (SEQ ID NO: 8) [ka] Ostreococcus tauri ATCC22665 BioDA protein (SEQ ID NO: 9) [ka] Sunflower (Helianthus annuus) BioDA protein (SEQ ID NO: 10) [ka] Quercus robur BioDA protein (SEQ ID NO: 11) [ka] Thraustotheca clavata BioDA protein (SEQ ID NO: 12) [ka] Schizosaccharomyces japonicus BioDA protein (SEQ ID NO: 13) [ka] Gibberella zeae BioDA protein (SEQ ID NO: 14) [ka] Barley (Hordeum vulgare) BIO3-BIO1 (SEQ ID NO: 15) [ka] Brassica napus BIO3-BIO1 (SEQ ID NO: 16) [ka] Cotton (Gossypium hirsutum) BIO3-BIO1 (SEQ ID NO: 17) [ka] Rice (Oryza sativa) BIO3-BIO1 (SEQ ID NO: 18) [ka] Soybean (Glycine max) BIO3-BIO1 (SEQ ID NO: 19) [ka] Wheat (Triticum aestivum) BIO3-BIO1 (SEQ ID NO: 20) [ka] BIODA / BIO3-BIO1 protein sequence-MTP removal Arabidopsis thaliana BioDAΔMTP protein (SEQ ID NO: 21) [ka] Zea mays BioDAΔMTP protein sequence (SEQ ID NO: 22) [ka] Taxus chinensis BioDA ΔMTP (SEQ ID NO: 23) [ka] Physcomitrium patens BioDA ΔMTP (SEQ ID NO: 24) [ka] Foxtail millet (Setaria italica) BioDA ΔMTP (SEQ ID NO: 25) [ka] Date palm (Phoenix dactylifera) BioDA ΔMTP (SEQ ID NO: 26) [ka] Ostreococcus tauri ATCC 22665 BioDA ΔMTP (SEQ ID NO: 27) [ka] Sunflower (Helianthus annuus) BioDA ΔMTP (SEQ ID NO: 28) [ka] Quercus robur BioDA ΔMTP protein (SEQ ID NO: 29) [ka] Thraustotheca clavata BioDA ΔMTP (SEQ ID NO: 30) [ka] Gibberella zeae BioDA ΔMTP (SEQ ID NO: 31) [ka] Barley (Hordeum vulgare) BIO3-BIO1ΔMTP (SEQ ID NO: 32) [ka] Brassica napus BIO3-BIO1 ΔMTP (SEQ ID NO: 33) [ka] Cotton (Gossypium hirsutum) BIO3-BIO1 ΔMTP (SEQ ID NO: 34) [ka] Rice (Oryza sativa) BIO3-BIO1ΔMTP (SEQ ID NO: 35) [ka] Soybean (Glycine max) BIO3-BIO1 ΔMTP (SEQ ID NO: 36) [ka] Wheat (Triticum aestivum) BIO3-BIO1ΔMTP (SEQ ID NO: 37) [ka] pET-24 His-AtBIO3-BIO1 E. coli expression vector (SEQ ID NO: 38) [ka] [ka] [ka] [ka]

Claims

1. 1. An in vitro method for determining whether a candidate compound is an inhibitor of diaminopelargonic acid aminotransferase (DAPA-AT), comprising: (i) assaying the candidate compound in an assay mixture comprising (a) the DAPA-AT, (b) an amine donor, (c) pyridoxal 5'-phosphate (PLP), and (d) 7-keto-8-aminopelargonic acid (KAPA); and (ii) detecting inhibition of the DAPA-AT by the candidate compound, wherein the amine donor comprises an aliphatic diamine or aliphatic polyamine, or a carbamate derivative thereof.

2. The method of claim 1, wherein the DAPA-AT is plant BIO1 or a functional derivative or fragment thereof.

3. The method of claim 1 or 2, wherein the DAPA-AT is provided as a bifunctional BIO3-BIO1 enzyme or a functional derivative or fragment thereof.

4. The method of any one of claims 1 to 3, wherein the DAPA-AT is Arabidopsis BIO3-BIO1 or a functional derivative or fragment thereof.

5. The method of claim 3, wherein the DAPA-AT is SEQ ID NO: 21 or a functional derivative or fragment thereof.

6. The method of any one of claims 1 to 5, wherein the DAPA-AT is recombinantly produced.

7. The amine donor has the formula (I): 【Chemistry 1】 Compounds of (Wherein, X is NH or CH 2 and R 1 is hydrogen or -[CH 2 ]pNH 2 and m and n are independently selected from 0, 1, 2, 3, or 4; and p=1, 2, 3 or 4) or R 1 The method of any one of claims 1 to 6, wherein is a carbamate derivative thereof, wherein is -C(O)OH.

8. 8. The method of claim 1, wherein the amine donor is selected from the group consisting of cadaverine, putrescine, spermidine, and spermine.

9. 9. The method of claim 8, wherein the amine donor is spermidine.

10. The method of any one of claims 1 to 9, wherein detecting the inhibition comprises monitoring the formation of diaminopelargonic acid (DAPA).

11. 11. The method of any one of claims 1 to 10, wherein the method utilizes an assay mixture, wherein the candidate compound is first incubated with the DAPA-AT (a) and PLP (c) in an enzyme mixture, and second, the enzyme mixture is combined with a substrate mixture comprising the amine donor (b) and the 7-keto-8-aminopelargonic acid (KAPA) (d).

12. The method of any one of claims 1 to 11, wherein the formation of DAPA is monitored by converting the DAPA into a fluorescent derivative.

13. The method of claim 12, wherein the fluorescent derivative is DAPA:o-phthalaldehyde.

14. The assay mixture comprises: (e) ATP and (f) CO 2 4. The method of claim 3, further comprising: detecting said inhibition by monitoring the formation of DAPA and / or dethiobiotin (DTB).

15. Formula (I): 【Chemistry 2】 Compounds of (Wherein, X is NH or CH 2 and R 1 represents hydrogen, —C(O)OH and —[CH 2 ] p NH 2 selected from the group consisting of: m and n are independently selected from 0, 1, 2, 3, or 4; and p=1, 2, 3 or 4) or R 1 Use of an amine donor, including its carbamate derivative, wherein is —C(O)OH, in an in vitro diaminopelargonate aminotransferase (DAPA-AT) assay.

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