Interaction of (4-hydroxyphenyl)pyruvate dioxygenase with the specific inhibitor 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is a non-heme Fe(II) enzyme that catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of the tyrosine catabolism pathway. Inhibition of HPPD by the triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC) is used to treat type I tyrosinemia, a rare but fatal defect in tyrosine catabolism. Although triketones have been used for many years as HPPD inhibitors for both medical and herbicidal purposes, the mechanism of inhibition is not well understood. The following work provides mechanistic insight into NTBC binding. The tautomeric population of NTBC in aqueous solution is dominated by a single enol as determined by NMR spectroscopy. NTBC preferentially binds to the complex of HPPD and FeII [HPPD.Fe(II)] as evidenced by a visible absorbance feature centered at 450 nm. The binding of NTBC to HPPD.Fe(II) was observed using a rapid mixing method and was shown to occur in two phases and comprise three steps. A hyperbolic dependence of the first observable process with NTBC concentration indicates a pre-equilibrium binding step followed by a limiting rate (K(1) = 1.25 +/- 0.08 mM, k(2) = 8.2 +/- 0.2 s(-1)), while the second phase (k(3) = 0.76 +/- 0.02 s(-1)) had no dependence on NTBC concentration. Neither K(1),k(2), nor k(3) was influenced by pH in the range of 6.0-8.0. Isotope effects on both k(2) and k(3) were observed when D(2)O is used as the solvent (for k(2), k(h)/k(d) = 1.3; for k(3), k(h)/k(d) = 3.2). It is therefore proposed that the bidentate association of NTBC with the active site metal ion (k(2)) precedes the Lewis acid-assisted conversion of the bound enol to the enolate (k(3)). Although the native enzyme without substrate reacts with molecular oxygen to form the oxidized holoenzyme, the HPPD.Fe(II).NTBC complex does not. When the complex is exposed to atmospheric oxygen, the absorbance feature associated with NTBC binding does not diminish over the course of 2 days. This means not only that the HPPD.Fe(II).NTBC complex does not oxidize but also that the dissociation rate constant for NTBC is essentially zero because any HPPD.Fe(II) that formed would readily oxidize in the presence of dioxygen. Consistent with this observation, EPR spectroscopy has shown that only 2% of the HPPD.Fe(II).NTBC complex forms an NO complex as compared to the holoenzyme.     

Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC

Di- and triketone inhibitors of (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are both effective herbicides and therapeutics. The inhibitory activity is used to halt the production of lipophilic redox cofactors in plants and also in humans to prevent accumulation of toxic metabolic byproducts that arise from specific inborn defects of tyrosine catabolism. The three-dimensional structure of the Fe(II) form of HPPD from Streptomyces avermitilis in complex with the inhibitor 2-[2-nitro-4-(triflouromethyl)benzoyl]-1,3-cyclohexanedione (NTBC) has been determined at a resolution of 2.5 A. NTBC coordinates to the active site metal ion, located at the bottom of a wide solvent-accessible cavity in the C-terminal domain of the protein. The iron is liganded in a predominantly five-coordinate, distorted square-pyramidal arrangement in which Glu349, His187, and His270 are protein-derived ligands and two other ligands are from the 5' and 7' oxygens of NTBC. There is a low-occupancy water molecule in the sixth coordination site in one of the protomers. The distance to His270 is unusually long at 2.5 A, and its orientation is somewhat distorted from ideal ligand geometry to within 2.8 A of the inhibitor nitro group. In contrast to the tetrameric quartenary structure observed for HPPD from other bacterial sources, the asymmetric unit is composed of two weakly associated protomers with a buried surface area of 1266 A(2) and a total of 12 hydrogen-bonding and no electrostatic interactions. The overall tertiary structure is similar to that of HPPD from Pseudomonas fluorescens (Serre et al., (1999) Structure 7, 977-988), although the position of the C-terminal alpha-helix is dramatically shifted. This C-terminal alpha-helix provides Phe364, which in combination with Phe336 sandwiches the phenyl ring of the bound NTBC; no other significant hydrogen-bonding or charge-pairing interactions are observed. Moreover, the structure reveals that, with the exception of Val189, NTBC makes contacts to only fully conserved amino acids. The combination of bidentate metal-ion coordination and pi-stacked aromatic rings is suggestive of a binding mode for the substrate and/or a transition state, which may be the origin of the exceedingly high affinity these inhibitors have for HPPD.     

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.     

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.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

3D-QSAR studies on 4-hydroxyphenylpyruvate dioxygenase inhibitors by comparative molecular field analysis (CoMFA)

A comparative molecular field analysis (CoMFA) of alkanoic acid 3-oxo-cyclohex-1-enyl ester and 2-acylcyclohexane-1,3-dione derivatives of 4-hydroxyphenylpyruvate dioxygenase inhibitors has been performed to determine the factors required for the activity of these compounds. The substrate's conformation abstracted from dynamic modeling of the enzyme-substrate complex was used to build the initial structures of the inhibitors. Satisfactory results were obtained after an all-space searching procedure, performing a leave-one out (LOO) cross-validation study with cross-validation q(2) and conventional r(2) values of 0.779 and 0.989, respectively. The results provide the tools for predicting the affinity of related compounds, and for guiding the design and synthesis of new HPPD ligands with predetermined affinities.     

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.     

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.     

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.     

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.     

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.     

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.     

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of β-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of β-triketone herbicides. The β-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified β-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I₅₀ values of 1.45, 0.96 and 11.5 μg mL⁻¹, respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O₂ consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I₅₀ values 15.0, 4.02, 3.14, 0.22 μg mL⁻¹ for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe²⁺ favors the binding of ligands with lipophilic moieties.     

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。     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

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.     

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.     

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.     

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.     

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.