Isolation of herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase from cultured Coptis japonica cells

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O(2). In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonica cells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC(50)) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.     

Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases

A high degree of selectivity toward the target site of the pest organism is a desirable attribute for new safer agrochemicals. To assist in the design of novel herbicides, we determined the crystal structures of the herbicidal target enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; EC 1.13.11.27) from the plant Arabidopsis thaliana with and without an herbicidal benzoylpyrazole inhibitor that potently inhibits both plant and mammalian HPPDs. We also determined the structure of a mammalian (rat) HPPD in complex with the same nonselective inhibitor. From a screening campaign of over 1000 HPPD inhibitors, six highly plant-selective inhibitors were found. One of these had remarkable (>1600-fold) selectivity toward the plant enzyme and was cocrystallized with Arabidopsis HPPD. Detailed comparisons of the plant and mammalian HPPD-ligand structures suggest a structural basis for the high degree of plant selectivity of certain HPPD inhibitors and point to design strategies to obtain potent and selective inhibitors of plant HPPD as agrochemical leads.     

Isolation of Herbicide-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Cultured Coptis japonica Cells

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O₂. In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonica cells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC₅₀) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.     

4-Hydroxyphenylpyruvate dioxygenase

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an Fe(II)-dependent, non-heme oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. This reaction involves decarboxylation, substituent migration and aromatic oxygenation in a single catalytic cycle. HPPD is a member of the alpha-keto acid dependent oxygenases that typically require an alpha-keto acid (almost exclusively alpha-ketoglutarate) and molecular oxygen to either oxygenate or oxidize a third molecule. As an exception in this class of enzymes HPPD has only two substrates, does not use alpha-ketoglutarate, and incorporates both atoms of dioxygen into the aromatic product, homogentisate. The tertiary structure of the enzyme would suggest that its mechanism converged with that of other alpha-keto acid enzymes from an extradiol dioxygenase progenitor. The transformation catalyzed by HPPD has both agricultural and therapeutic significance. HPPD catalyzes the second step in the pathway for the catabolism of tyrosine, that is common to essentially all aerobic forms of life. In plants this pathway has an anabolic branch from homogentisate that forms essential isoprenoid redox cofactors such as plastoquinone and tocopherol. Naturally occurring multi-ketone molecules act as allelopathic agents by inhibiting HPPD and preventing the production of homogentisate and hence required redox cofactors. This has been the basis for the development of a range of very effective herbicides that are currently used commercially. In humans, deficiencies of specific enzymes of the tyrosine catabolism pathway give rise to a number of severe metabolic disorders. Interestingly, HPPD inhibitor/herbicide molecules act also as therapeutic agents for a number of debilitating and lethal inborn defects in tyrosine catabolism by preventing the accumulation of toxic metabolites.     

4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.     

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway.

BACKGROUND: In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants. Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism. RESULTS: We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%). The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC). Each structural domain contains two repeated betaalpha betabeta betaalpha modules. There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule. CONCLUSIONS: The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes. These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom. This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction.     

Two roads diverged: the structure of hydroxymandelate synthase from Amycolatopsis orientalis in complex with 4-hydroxymandelate

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.     

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol–gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.     

Evidence for the mechanism of hydroxylation by 4-hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase from intermediate partitioning in active site variants

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.     

Catalytic, noncatalytic, and inhibitory phenomena: kinetic analysis of (4-hydroxyphenyl)pyruvate dioxygenase from Arabidopsis thaliana

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) incorporates both atoms of molecular oxygen into 4-hydroxyphenylpyruvate (HPP) to form homogentisate (HG). This reaction has direct relevance in both medicine and agriculture. In humans, the specific inhibition of HPPD alleviates the symptoms of diseases that arise from tyrosine catabolism defects. However, in plants, the inhibition of HPPD bleaches, stunts, and ultimately kills the organism. The reason for this is that in mammalian metabolism the product HG does not feed into other pathways, whereas in plants it is the precursor for the redox active portion of tocopherols and plastoquinones. There are a number of commercially available herbicides that directly target the inhibition of the HPPD reaction. Plant HPPD however is largely uncharacterized in terms of its catalysis and inhibition reactions. In this study, we examine the catalysis and inhibition of HPPD from Arabidopsis thaliana (AtHPPD). We have expressed AtHPPD and purified the enzyme to high specific activity. This form of HPPD accumulates two transient species in single turnover reactions with the native substrate HPP. These transients appear to be equivalent to intermediates I and III observed in the enzyme from Streptomyces (Johnson-Winters et al. (2005), Biochemistry, 44, 7189-7199). The first intermediate is a relatively strongly absorbing species with maxima at 380 and 490 nm. This species decays to a second intermediate that is fluorescent and has been assigned as the complex of the enzyme with the product, HG. The decay of this intermediate is rate-determining in multiple turnover reactions. The reaction of the enzyme with the analogue of the substrate, phenylpyruvate (PPA), is noncatalytic. A single turnover reaction is observed with this ligand that renders the enzyme oxidized to the ferric form, consumes a stoichiometric amount of dioxygen, and yields 66% phenylacetate as a product. Additional absorbance features at 365 and 670 nm accumulate during inactivation and give the inactivated enzyme a green color but has the same molecular mass as the active enzyme as determined by mass spectrometry.     

新型白化型除草剂靶标酶对羟苯基丙酮酸双加氧酶及其耐性转基因植物研究进展*

对羟苯基丙酮酸双加氧酶(ρ-hydroxyphenylpyruvate dioxygenase,HPPD;EC 1.13.11.27)催化生物体内对羟苯基丙酮酸与O2作用形成尿黑酸的反应,是植物体中质体醌和生育酚生物合成途径的关键酶。当其活性受到抑制时,植物体中作为类胡萝卜素生物合成途径中最终电子受体和光合链电子传递体的质体醌的生物合成受阻,进而导致类胡萝卜素合成减少,光合链电子传递受阻,致使植物体出现白化症状。目前已经开发了多种以HPPD为靶标的除草剂,该类除草剂及抗除草剂转基因植物研究具有广阔的前景。对这一新型白化型除草剂靶标酶以及耐该类除草剂转基因植物的研究进展作了简要综述。     

Overexpression of the enzyme p-hydroxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisic acid (HGA), the aromatic precursor for the biosynthesis of vitamin E (α-tocopherol) and plastoquinone. In order to determine if increased HPPD activity could positively impact tocopherol yields, transgenic plants were generated that overexpressed the gene encoding Arabidopsis HPPD. Transgenic plants exhibiting high levels of HPPD expression were identified by increased tolerance to a competitive inhibitor of HPPD, the herbicide sulcotrione. HPPD gene expression in these transgenic lines, as determined at the RNA, protein and activity levels, was at least 10-fold higher than that of wild-type plants. Subsequent tocopherol analysis of leaf and seed material revealed that the increased HPPD expression resulted in up to a 37% increase in leaf tocopherol levels and a 28% increase in seed tocopherol levels relative to control plants. These results demonstrate that HPPD activity, and likely HGA levels, are at least one factor limiting the production of tocopherols in photosynthetic and non-photosynthetic plant tissues.     

Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT‐independent pathway for tocotrienol synthesis has also been shown to occur by de‐regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co‐expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi‐functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2–1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA‐expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA‐induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT‐catalyzed tocotrienol synthesis.     

Inhibition of p-hydroxyphenylpyruvate dioxygenase by the diketonitrile of isoxaflutole: a case of half-site reactivity

p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from p-hydroxyphenylpyruvate and molecular oxygen. In plants, this enzyme is the molecular target of new families of very active bleaching herbicides. In the study presented here, we report for the first time on the purification to homogeneity of a plant enzyme, as obtained from recombinant Escherichia coli cells expressing a cDNA encoding carrot HPPD. The purified enzyme allowed us to carry out a detailed characterization of the inhibitory properties of a diketonitile (DKN), the active inhibitor formed from the benzoylisoxazole herbicide isoxaflutole. Inhibition kinetic analyses confirmed that DKN exerts a slow and tight-binding inhibition of HPPD, competitive with respect to the p-hydroxyphenylpyruvate substrate. The stoichiometry of DKN binding to HPPD determined by kinetic analyses or by direct binding of [(14)C]DKN revealed a half-site reactivity of DKN.     

Accumulation of multiple intermediates in the catalytic cycle of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation in a single catalytic cycle. HPPD is a unique member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate or oxidize an organic molecule. We have examined the reaction coordinate of HPPD from Streptomyces avermitilis using rapid mixing pre-steady-state methods in conjunction with steady-state kinetic analyses. Acid quench reactions and product analysis of homogentisate indicate that HPPD as isolated is fully active and that experiments limited in dioxygen concentration with respect to that of the enzyme do involve a single turnover. These experiments indicate that during the course of one turnover the concentration of homogentisate is stoichiometric with enzyme concentration by approximately 200 ms, well before the completion of the catalytic cycle. Subsequent single turnover reactions were monitored spectrophotometrically under pseudo-first-order and matched concentration reactant conditions. Three spectrophotometrically distinct intermediates are observed to accumulate. The first of these is a relatively strongly absorbing species with maxima at 380 and 480 nm that forms with a rate constant (k(1)) of 7.4 x 10(4) M(-)(1) s(-)(1) and then decays to a second intermediate with a rate constant (k(2)) of 74 s(-)(1). The rate constant for the decay of the second intermediate (k(3)) is 13 s(-)(1) and is concomitant with the formation of the product, homogentisate, based on rapid quench and pre-steady-state fluorescence measurements. The rate constant for this process decreases to 7.6 s(-)(1) when deuterons are substituted for protons in the aromatic ring of the substrate. The release of product from the enzyme is rate limiting and occurs at 1.6 s(-)(1). This final event exhibits a kinetic isotope effect of 2 with deuterium oxide as the solvent, consistent with a solvent isotope effect on V(max) of 2.6 observed in steady-state experiments.     

Free energy calculations elucidate substrate binding,gating mechanism,and tolerance‐promoting mutations in herbicide target 4‐hydroxyphenylpyruvate dioxygenase

4‐Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide‐tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C‐terminal helix H11 in HPPD. We compared gating equilibria in Arabidopsis thaliana (At) and Zea mays (Zm) wild‐type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open–closed transition and discovered a ligand‐mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in At mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new At mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.     

FUNCTIONAL CHARACTERIZATION AND EXPRESSION ANALYSIS OF p‐HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The enzyme p‐hydroxyphenylpyruvate dioxygenase (HPPD) is very important in prenylquinone biosynthesis in all photosynthetic organisms. In this study, we present the functional characterization and expression analysis of HPPD from the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. Recombinant HPPD1 enzyme was purified and characterized. Kinetic analysis revealed a K_m of 49 μM for p‐hydroxyphenylpyruvate, similar to other HPPDs. The size of HPPD subunit was estimated as 47 kDa by SDS‐PAGE, in accordance with the predicted molecular size after HPPD1 cDNA sequence. However, native HPPD1 enzyme showed an apparent molecular mass of 188 kDa and a homotetrameric structure, which suggests a reconsideration of the idea that all eukaryotic HPPDs have a homodimeric structure while all prokaryotic HPPDs are homotetramers. Expression analysis by Northern blot revealed that hppd1 expression is strongly up‐regulated by low temperature and poorly regulated by high temperature, darkness, or moderate light changes, suggesting that Chlamydomonas HPPD may play an important role in the synthesis of tocopherols and/or plastoquinones under stress conditions in the physiological context of the adaptation to growth at low temperatures.     

The Interaction of Hydroxymandelate Synthase with the 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor: NTBC

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of polycyclic non-ribosomal peptide antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O₂, and also conducts a dioxygenation reaction. The difference between the two lies in the insertion of the second oxygen atom, HMS directing this atom onto the benzylic carbon of the substrate while HPPD hydroxylates the aromatic C1 carbon. We have shown that HMS will bind NTBC, a herbicide/therapeutic whose mode of action is based on the inhibition of HPPD. This occurs despite residue differences at the active site of HMS from those known to contact the inhibitor in HPPD. Moreover, the minimal kinetic mechanism for association of NTBC to HMS differs only slightly from that observed with HPPD. The primary difference is that three charge-transfer species are observed to accumulate during association. The first reversible complex forms with a weak dissociation constant of 520 μM, the subsequent two charge-transfer complexes form with rate constants of 2.7 s⁻¹ and 0.67 s⁻¹. As was the case for HPPD, the final complex has the most intense charge-transfer, is not observed to dissociate, and is unreactive towards dioxygen.     

Transgenic rice grains expressing a heterologous ρ-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels

We generated transgenic rice plants overexpressing Arabidopsis thaliana ρ-hydroxyphenylpyruvate dioxygenase (HPPD), which catalyzes the first committed step in vitamin E biosynthesis. Transgenic grains accumulated marginally higher levels of total tocochromanols than controls, reflecting a small increase in absolute tocotrienol synthesis (but no change in the relative abundance of the α and γ isoforms). In contrast, there was no change in the absolute tocopherol level, but a significant shift from the γ to the α isoform. These data confirm HPPD is not rate limiting, and that increasing flux through the early pathway reveals downstream bottlenecks that act as metabolic tipping points.