Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens.

Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins labeled with stable isotopes or selenomethionine is essential for the determination of protein structures. A completely defined medium for the expression and stable isotope labeling of proteins in P. fluorescens has been developed. The expression level of Bacillus thuringiensis Cry34 in the modified medium is comparable to that obtained in the original medium. In addition, more than 95% incorporation of 15N was obtained in Cry34 using 15N ammonium sulfate and the quality of the protein, as assessed by NMR analysis, is comparable to that made using commercial medium. High levels of selenomethionine (SeMet) incorporation in the Xenorhabdus nematophilus insecticidal protein XptA2 were also obtained in P. fluorescens using the defined medium, allowing development of a method for obtaining highly purified XptA2. The following observations were made when inhibitors of endogenous methionine biosynthesis were used in P. fluorescens culture when SeMet was substituted in XptA2: (I) there is little inhibition of cell growth or recombinant XptA2 expression in the presence of SeMet concentrations up to 300 mg/L in cell culture, (II) there was greater than 95% SeMet incorporation ratio in recombinant SeMet-labeled XptA2 (SeMet-XptA2) and the incorporation ratio is consistent and reproducible and (III) finally, purified SeMet-XptA2 possesses similar protein structure and insecticidal activity relative to the unlabeled counterpart XptA2 as shown by bioassay and differential scanning calorimetric analysis. The high SeMet incorporation should provide high accuracy and resolution in XptA2 phase determination by multiwavelength anomalous diffraction (MAD), indicating that P. fluorescens is an excellent expression host to produce SeMet-labeled proteins for structural study.

Characterization of two hydroxytrichloropicolinic acids: application of the one-bond chlorine-isotope effect in 13C NMR.

The structures of 4-hydroxy-3,5,6-trichloropyridine-2-carboxylic acid (1a) and 6-hydroxy-3,4,5-trichloro-2-carboxylic acid (1b) were verified by the NMR analysis of their corresponding methylated and decarboxylated derivatives 2,3,5-trichloro-4-methoxypyridine (5) and 3,4,5-trichloro-2-methoxypyridine (8), respectively. The 6-hydroxy isomer (1a) was found to be in equilibrium with its pyridinone tautomer as evidenced by the formation of significant amounts of 3,4,5-trichloro-1-methyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid methyl ester (6b) on exhaustive methylation. The one-bond chlorine-isotope effect was used and shown to be an effective tool for the identification of chlorinated carbons in (13)C NMR spectra providing an additional tool for solving structural problems in chlorinated compounds.

Improved synthetic route to quinoxyfen photometabolite 2-chloro-10-fluorochromeno[2,3,4-de]quinoline.

BACKGROUND: Quinoxyfen is a fungicide developed by Dow AgroSciences for the control of powdery mildew. Re-registration studies required gram quantities of 2-chloro-10-fluorochromeno[2,3,4-de]quinoline, a photometabolite of quinoxyfen. The only previous method of preparation of this photometabolite was by photolysis of quinoxyfen in less than 1% yield. Therefore, a new method allowing for the preparation of this photometabolite in gram quantities was required. RESULTS: Several different metal catalyzed intramolecular cyclization approaches were investigated for the synthesis of 2-chloro-10-fluorochromeno[2,3,4-de]quinoline. While most methods failed to provide the desired product from a 2-bromophenyl derivative of quinoxyfen, a novel one-pot two-step synthesis led to the desired material in good yield from quinoxyfen. CONCLUSION: A short and efficient synthetic route was developed to access 2-chloro-10-fluorochromeno[2,3,4-de]quinoline from readily available (4-fluoro-2-hydroxyphenyl)boronic acid and quinoxyfen and was found to be scalable, which enabled the preparation of the desired photometabolite in gram quantities thus meeting material requirements to complete regulatory studies for the re-registration of quinoxyfen. © 2017 Society of Chemical Industry.

Uptake, translocation and metabolism of the herbicide florasulam in wheat and broadleaf weeds.

Florasulam is a triazolopyrimidine sulfonanilide post-emergence broadleaf herbicide for use in wheat (Triticum aestivum L.). The selectivity of florasulam to wheat has been determined to be related primarily to a differential rate of metabolism between wheat with a half-life of 2.4 h and broadleaf weeds with half-lives ranging from 19 to >48 h. To a lesser extent, selectivity, at least for the broadleaf weed cleavers (Galium aparine L.), involves uptake differences. Rate of metabolism data were generated using greenhouse-grown plants injected with radiolabelled florasulam and subsequent extraction and processing by high-performance liquid chromatography (HPLC). Structures of metabolites were determined by isolation for nuclear magnetic resonance and liquid chromatography/mass spectrometry. Wheat plants metabolised florasulam by hydroxylation of the aniline ring para to the nitrogen, followed by conjugation to glucose. Metabolism by broadleaf weeds was so slow that isolation of metabolite was not possible, but comparison of HPLC data suggested hydroxylation as the major pathway.

Metabolism of strobilurins by wheat cell suspension cultures.

Strobilurin fungicides are a leading class of antifungal chemicals used today in agricultural applications. Although degradation of some strobilurin fungicides has been assessed in plant residues, little information has appeared in the literature concerning the rates of metabolism of these fungicides in plants. In this study, we explored plant metabolism of three strobilurin fungicides, azoxystrobin, kresoxim-methyl, and trifloxystrobin, using wheat cell suspension cultures. Trifloxystrobin and kresoxim-methyl were completely metabolized within 24 h, whereas the metabolism of azoxystrobin was relatively slow with half-lives up to 48 h depending on specific experimental conditions. Metabolic rates of these fungicides were affected by the amounts of compound and cells added to the media. Structural analysis of metabolites of trifloxystrobin and kresoxim-methyl by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR) indicated that trifloxystrobin was first demethylated followed by subsequent hydroxylation, whereas kresoxim-methyl was largely demethylated. In contrast, a number of minor metabolites of azoxystrobin were present suggesting a differential metabolism of strobilurins by wheat cells.

The impact of uptake, translocation and metabolism on the differential selectivity between blackgrass and wheat for the herbicide pyroxsulam.

BACKGROUND: Wheat shows selectivity to pyroxsulam, a new broad-spectrum herbicide with high activity on blackgrass. Studies were performed to establish whether uptake, translocation or metabolism were responsible for the differential activity in wheat compared with blackgrass. In addition, the effect of the safener cloquintocet-mexyl on metabolism was evaluated in wheat and blackgrass shoots. RESULTS: Root uptake of pyroxsulam in blackgrass was significantly higher than in wheat, suggesting a possible activity enhancement in blackgrass owing to root uptake. Translocation to foliage from root uptake as well as translocation out of treated foliage following foliar applications was low in wheat compared with blackgrass, likely owing to the rapid metabolism of pyroxsulam in wheat. Wheat metabolized pyroxsulam significantly faster than blackgrass to the less active O-dealkylation product. Wheat shoots metabolized pyroxsulam faster when the safener cloquintocet-mexyl was present, but cloquintocet-mexyl did not increase the rate of metabolism in blackgrass. CONCLUSIONS: The selectivity of pyroxsulam to wheat relative to blackgrass was connected primarily with differences in the rate of metabolism and generation of an inactive metabolite. Metabolism in wheat restricted subsequent movement of radioactivity out of the treated leaf. The rapid metabolism in wheat was increased by the addition of cloquintocet-mexyl.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase from maize: molecular and biochemical characterization.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase, an enzyme encoded by the gene IPK1, catalyzes the terminal step in the phytic acid biosynthetic pathway. We report here the isolation and characterization of IPK1 cDNA and genomic clones from maize (Zea mays). DNA Southern-blot analysis revealed that ZmIPK1 in the maize genome constitutes a small gene family with two members. Two nearly identical ZmIPK1 paralogs, designated as ZmIPK1A and ZmIPK1B, were identified. The transcripts of ZmIPK1A were detected in various maize tissues, including leaves, silks, immature ears, seeds at 12 d after pollination, midstage endosperm, and maturing embryos. However, the transcripts of ZmIPK1B were exclusively detected in roots. A variety of alternative splicing products of ZmIPK1A were discovered in maize leaves and seeds. These products are derived from alternative acceptor sites, alternative donor sites, and retained introns in the transcripts. Consequently, up to 50% of the ZmIPK1A transcripts in maize seeds and leaves have an interrupted open reading frame. In contrast, only one type of splicing product of ZmIPK1B was detected in roots. When expressed in Escherichia coli and subsequently purified, the ZmIPK1 enzyme catalyzes the conversion of myo-inositol 1,3,4,5,6-pentakisphosphate to phytic acid. In addition, it is also capable of catalyzing the phosphorylation of myo-inositol 1,4,6-trisphosphate, myo-inositol 1,4,5,6-tetrakisphosphate, and myo-inositol 3,4,5,6-tetrakisphosphate. Nuclear magnetic resonance spectroscopy analysis indicates that the phosphorylation product of myo-inositol 1,4,6-trisphosphate is inositol 1,2,4,6-tetrakisphosphate. Kinetic studies showed that the K(m) for ZmIPK1 using myo-inositol 1,3,4,5,6-pentakisphosphate as a substrate is 119 microm with a V(max) at 625 nmol/min/mg. These data describing the tissue-specific accumulation and alternative splicing of the transcripts from two nearly identical ZmIPK1 paralogs suggest that maize has a highly sophisticated regulatory mechanism controlling phytic acid biosynthesis.