Insights into 4-hydroxyphenylpyruvate dioxygenase-inhibitor interactions from comparative structural biology.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a key role in tyrosine metabolism and has been identified as a promising target for herbicide and drug discovery. The structures of HPPD complexed with different types of inhibitors have been determined previously. We summarize the structures of HPPD complexed with structurally diverse molecules, including inhibitors, natural products, substrates, and catalytic intermediates; from these structures, the detailed inhibitory mechanisms of different inhibitors were analyzed and compared, and the key structural factors determining the slow-binding behavior of inhibitors were identified. Further, we propose four subpockets that accommodate different inhibitor substructures. We believe that these analyses will facilitate in-depth understanding of the enzymatic reaction mechanism and enable the design of new inhibitors with higher potency and selectivity.

The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.

Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa.

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to ß-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying ß-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Species differences and human relevance of the toxicity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and a new approach method in vitro for investigation.

The mode of action (MoA) of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides in mammals is well described and is generally accepted to be due to a build-up of excess systemic tyrosine which is associated with the range of adverse effects reported in laboratory animals. What is less well accepted is the basis for the marked difference in the effects of HPPD inhibitors that has been observed across experimental species and humans, where some species show significant toxicities whereas in other species exposure causes few effects. The activity of the catabolic enzyme tyrosine aminotransferase (TAT) varies across species including humans and it is hypothesized that this primarily accounts for the different levels of tyrosinemia observed between species and leads to the subsequent differences in toxicity. The previously reported activities of TAT in different species showed large variation, were inconsistent, have methodological uncertainties and could lead to a reasonable challenge to the scientific basis for the species difference in response. To provide clarity, a new method was developed for the simultaneous and systematic measurement of TAT in vitro using robust methodologies in a range of mammalian species including human. The results obtained showed general correlation between high TAT activity and low in vivo toxicity when using a model based on hepatic cytosol and a very convincing correlation when using a primary hepatocyte model. These data fully support the role of TAT in explaining the species differences in toxicity. Moreover, this information should give greater confidence in selecting the most appropriate animal model (the mouse) for human health risk assessment and for key classification and labeling decision-making.

Multivariate image analysis applied to quantitative structure-activity relationships and docking studies of recent hydroxyphenylpyruvate deoxygenase inhibitors.

BACKGROUND: Mesotrione is a triketone widely used as an inhibitor of the hydroxyphenylpyruvate deoxygenase (HPPD) enzyme. However, new agrochemicals should be developed continuously to tackle the problem of herbicide resistance. Two sets of mesotrione analogs have been synthesized recently and they have demonstrated successful phytotoxicity against weeds. In this study, these compounds were joined to form a single data set and the HPPD inhibition of this enlarged library of triketones was modeled using multivariate image analysis applied to quantitative structure-activity relationships (MIA-QSAR). Docking studies were also carried out to validate the MIA-QSAR findings and to aid the interpretation of ligand-enzyme interactions responsible for the bioactivity (pIC50 ). RESULTS: The MIA-QSAR models based on van der Waals radii (rvdW ), electronegativity (ε), and the rvdW /ε ratio as molecular descriptors were both predictive to an acceptable degree (r2 ≥ 0.80, q2 ≥ 0.68 and r2 pred ≥ 0.68). Subsequently, partial least squares (PLS) regression parameters were applied to predict the pIC50 values of newly proposed derivatives, yielding a few promising agrochemical candidates. The calculated log P for most of these derivatives was found to be higher than that of mesotrione and the library compounds, indicating that they should be less prone to leach out and contaminate groundwater. CONCLUSION: Multivariate image analysis descriptors corroborated by docking studies were capable of modeling the herbicidal activities of 68 triketones reliably. Due to the substituent effects at the triketone framework, particularly of a nitro group in R3 , promising analogs could be designed. The P9 proposal demonstrated higher calculated activity and log P than commercial mesotrione. © 2023 Society of Chemical Industry.

Identification of 4-hydroxyphenylpyruvate dioxygenase inhibitors by virtual screening, molecular docking, molecular dynamic simulation.

BACKGROUND: 4-Hydroxyphenylpyruvate dioxygenase (HPPD) herbicides control broadleaf and gramineous weeds with better crop safety for corn, sorghum and wheat. Multiple screening models in silico have been established to obtain novel lead compounds as HPPD inhibition herbicides. RESULTS: Topomer comparative molecular field analysis (CoMFA) combined with topomer search technology and Bayesian, genetic approximation functions (GFA) and multiple linear regression (MLR) models generated by calculating different descriptors were constructed for the quinazolindione derivatives of HPPD inhibitors. The coefficient of determination (r2 ) of topomer CoMFA, MLR and GFA were 0.975, 0.970 and 0.968, respectively; all the models established displayed excellent accuracy and high predictive capacity. Five compounds with potential HPPD inhibition were obtained via screening fragment library combined with the validation of the above models and molecular docking studies. After molecular dynamics (MD) validation and absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction, the compound 2-(2-amino-4-(4H-1,2,4-triazol-4-yl) benzoyl)-3-hydroxycyclohex-2-en-1-one not only exhibited stable interactions with the protein but also high solubility and low toxicity, and has potential as a novel HPPD inhibition herbicide. CONCLUSION: In this study, five compounds were obtained through multiple quantitative structure-activity relationship screening. Molecular docking and MD experiments showed that the constructed approach had good screening ability for HPPD inhibitors. This work provided molecular structural information for developing novel, highly efficient and low-toxicity HPPD inhibitors. © 2023 Society of Chemical Industry.

Inhibition of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis.

Unlike the indispensable function of the steroid hormone brassinosteroid (BR) in regulating plant growth and development, the metabolism of secondary metabolites regulated by BR is not well known. Here we show that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated higher content of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid content. We demonstrated that BR transcriptionally suppresses 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. We found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the promoter of HPPD, and HPPD suppression by BR was increased in the bzr1-1D gain-of-function mutation. Interestingly, dark-induced HPPD expression did not cause carotenoid accumulation, due to down-regulation of other carotenoid biosynthetic genes in the dark. Our results suggest that BR regulates different physiological responses in dark and light through inhibition of HPPD expression.

Melanin biopolymer synthesis using a new melanogenic strain of Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing 4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi.

BACKGROUND: Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report the high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD). RESULTS: Melanin synthesis of F. kingsejongi strain was confirmed via melanin synthesis inhibition test, melanin solubility test, genome analysis, and structural analysis of purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6 × His-tagged and native forms of HPPD. The specific activity of F. kingsejongi HPPD was 1.2 ± 0.03 µmol homogentisate/min/mg-protein. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h. CONCLUSIONS: Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large-scale bacterial melanin production. Furthermore, F. kingsejongi strain could serve as a model to elucidate the regulation of melanin biosynthesis pathway and its networks with other cellular pathways, and to understand the cellular responses of melanin-producing bacteria to environmental changes, including nutrient starvation and other stresses.

Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Comparison of the binding interactions of 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicides with humic acid: Insights from multispectroscopic techniques, DFT and 2D-COS-FTIR.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor is one of the important herbicides to solve the problem of weed control. With the widespread and continued use of HPPD inhibitor (HPPDi) herbicides, it may inevitably put pressure on the environment. Humic acid (HA) can effectively interact with pesticides through sorption or covalent bond formation and promote the degradation of pesticides, which can reduce the risk of pesticides in the environment. In the present study, the interactions of four HPPDi herbicides (sulcotrione, tembotrione, topramezone and mesotrione) with HA were reported and comparative assessment of the binding using multispectral technology, density functional theory (DFT) calculation and two-dimensional correlation spectroscopy (2D-COS). Time-resolved measurements and the Stern-Volmer constant at different temperature verified that HPPDi can bind with HA through the static quenching mechanism. From the thermodynamic parameters, the interaction force between HA and sulcotrione, tembotrione, topramezone and mesotrione was provided by electrostatic force. DFT, binding constant and three-dimensional (3D) fluorescence peak variation all indicated that the order of the binding ability of the four HPPDi and HA was mesotrione > tembotrione > sulcotrione > topramezone. According to dynamic light scattering (DLS), pH 7 is most conducive to the formation of HA-HPPDi complexes. Fourier transform infrared spectroscopy (FTIR) and 2D-COS showed that HA combined with HPPDi through aromatic C-H, CO and C-X, and the first binding group to HA was almost all CO. Sulcotrione, tembotrione, topramezone and mesotrione quench the endogenous fluorescence of HA by a static quenching mechanism and bind to HA through electrostatic interaction to form a complex. These results provide important insights into the combination of environmental pollutants with HA.

Using RNA-seq to characterize responses to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide resistance in waterhemp (Amaranthus tuberculatus).

BACKGROUND: Waterhemp (Amaranthus tuberculatus (Moq.) J.D. Sauer) is a problem weed commonly found in the Midwestern United States that can cause crippling yield losses for both maize (Zea mays L.) and soybean (Glycine max L. Merr). In 2011, 4-hydroxyphenylpyruvate-dioxygenase (HPPD, EC 1.13.11.27) inhibitor herbicide resistance was first reported in two waterhemp populations. Since the discovery of HPPD-herbicide resistance, studies have identified the mechanism of resistance and described the inheritance of the herbicide resistance. However, no studies have examined genome-wide gene expression changes in response to herbicide treatment in herbicide resistant and susceptible waterhemp. RESULTS: We conducted RNA-sequencing (RNA-seq) analyses of two waterhemp populations (HPPD-herbicide resistant and susceptible), from herbicide-treated and mock-treated leaf samples at three, six, twelve, and twenty-four hours after treatment (HAT). We performed a de novo transcriptome assembly using all sample sequences. Following assessments of our assembly, individual samples were mapped to the de novo transcriptome allowing us to identify transcripts specific to a genotype, herbicide treatment, or time point. Our results indicate that the response of HPPD-herbicide resistant and susceptible waterhemp genotypes to HPPD-inhibiting herbicide is rapid, established as soon as 3 hours after herbicide treatment. Further, there was little overlap in gene expression between resistant and susceptible genotypes, highlighting dynamic differences in response to herbicide treatment. In addition, we used stringent analytical methods to identify candidate single nucleotide polymorphisms (SNPs) that distinguish the resistant and susceptible genotypes. CONCLUSIONS: The waterhemp transcriptome, herbicide-responsive genes, and SNPs generated in this study provide valuable tools for future studies by numerous plant science communities. This collection of resources is essential to study and understand herbicide effects on gene expression in resistant and susceptible weeds. Understanding how herbicides impact gene expression could allow us to develop novel approaches for future herbicide development. Additionally, an increased understanding of the prolific traits intrinsic in weed success could lead to crop improvement.

4-Hydroxyphenylpyruvate Dioxygenase Thermolability Is Responsible for Temperature-Dependent Melanogenesis in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains.IMPORTANCEAeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates.

Characterization of 4-hydroxyphenylpyruvate dioxygenases, inhibition by herbicides and engineering for herbicide tolerance in crops.

4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with kcat/KmHPP values ranging from 0.8 to 2.5 s-1 µM-1. Most were also highly sensitive to herbicides with, for example, Ki values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, Ki value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (CallistoTM) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of Ki to KmHPP, by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre.

QUESTION: Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING: Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ±â€¯0.19) and three (0.15 ±â€¯0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ±â€¯0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ±â€¯0.08) and three (0.19 ±â€¯0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ±â€¯0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean.

By transgenic expression technology, a modified 4-hydroxyphenylpyruvate dioxygenase enzyme (HPPD W336) originating from Pseudomonas fluorescens is expressed in MST-FGØ72-2 soybean to confer tolerance to 4-benzoyl isoxazole and triketone type of herbicides. Characterization and safety assessment of HPPD W336 were performed. No relevant sequence homologies were found with known allergens or toxins. Although sequence identity to known toxins showed identity to HPPD proteins annotated as hemolysins, the absence of hemolytic activity of HPPD W336 was demonstrated in vitro. HPPD W336 degrades rapidly in simulated gastric fluid. The absence of toxicity and hemolytic potential of HPPD W336 was confirmed by in vivo studies. The substrate spectrum of HPPD W336 was compared with wild type HPPD proteins, demonstrating that its expression is unlikely to induce any metabolic shifts in soybean. The potential effect of expression of HPPD W336 on metabolic pathways related to tyrosine was investigated by comparing seed composition of MST-FGØ72-2 soybean with non-genetically modified varieties, demonstrating that expression of HPPD W336 does not change aromatic amino acid, homogentisate and tocochromanol levels. In conclusion, HPPD W336 was demonstrated to be as safe as other food proteins. No adverse metabolic effects were identified related to HPPD W336 expression in MST-FGØ72-2 soybean.

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

Synthesis and biological activity of benzoylcyclohexanedione herbicide SYP-9121.

Benzoylcyclohexanedione herbicides work by inhibiting 4-hydroxyphenylpyruvate dioxygenase which was the last new target site introduced for herbicides. In an attempt to find new 4-hydroxyphenylpyruvate dioxygenase inhibitors with high efficacy and selectivity, a novel benzoylcyclohexanedione compound SYP-9121 was synthesized and studied in greenhouse and field. In the greenhouse, SYP-9121 showed broad spectrum herbicidal activity and good safety to maize. Its control of barnyard grass, crabgrass, redroot pigweed, purslane, dayflower and night shade was equivalent to that of the commercial herbicide mesotrione. Three field trials in summer maize showed that SYP-9121 could efficiently control both grass and broadleaf weeds with good selectivity. Herbicidal activity of SYP-9121 was comparable to that of mesotrione.

Discovery of (2-benzoylethen-1-ol)-containing 1,2-benzothiazine derivatives as novel 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting-based herbicide lead compounds.

A series of (2-benzoylethen-1-ol)-containing benzothiazine derivatives was synthesized, and their herbicidal activities were first evaluated. The bioassay results indicated that some of 3-benzoyl-4-hydroxy-2-methyl-2H-1,2-benzothiazine-1,1-dioxide derivatives displayed good herbicidal activity in greenhouse testing, especially, compound 4w had good pre-emergent herbicidal activities against Brassica campestris, Amaranthus retroflexus and Echinochloa crusgalli even at a dosage of 187.5 g ha(-1). More importantly, compound 4w displayed significant inhibitory activity against Arabidopsis thaliana HPPD and was identified as the most potent candidate with IC50 value of 0.48 µM, which is better than the commercial herbicide sulctrione (IC50=0.53 µM) and comparable with the commercial herbicide mesotrione (IC50=0.25 µM). The structure-activity relationships was studied and provided some useful information for improving herbicidal activity. The present work indicated that (2-benzoylethen-1-ol)-containing 1,2-benzothiazine motif could be a potential lead structure for further development of novel HPPD inhibiting-based herbicides.

Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.