Inhibition of Barnyardgrass 4-Hydroxyphenylpyruvate Dioxygenase by Sulcotrione

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) was partially purified from barnyardgrass (Echinochloa crus-galli L.) leaves and assayed by high-performance liquid chromatography analysis of product formation or by the capture of released 14CO2. The bleaching herbicide sulcotrione [2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione] was shown to be a potent, linear competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase. Kinetic analyses determined that the Km for the substrate, 4-hydroxyphenylpyruvate, was 4.3 [mu]M, and the Ki value was 9.8 nM for sulcotrione.     

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.     

The role of metals in the enzymatic and nonenzymatic oxidation of epinephrine

The effects of transition metals on nonenzymatic and ceruloplasmin catalyzed epinephrine oxidation were investigated by studying rates of epinephrine oxidation in purified buffers and in the presence of metal chelating agents. We found that epinephrine does not “autoxidize” in sodium chloride solutions prepared with deionized water that was further purified by chromatography over Chelex 100 resin prior to use. Epinephrine was oxidized rapidly in sodium chloride prepared with tap water (1.20±0.12 nmoles/min) or in deionized water (0.40±0.80 nmoles/min), but this oxidation was prevented by the addition of Desferal, a potent metal chelating agent. Epinephrine oxidation was enhanced upon the addition of ceruloplasmin, and this oxidation rate could be slowed, but not eliminated, by the addition of Desferal. If epinephrine solutions were preincubated for 72 hours with Desferal prior to ceruloplasmin addition, however, no oxidation was observed. Epinephrine was shown to form colored complexes with both iron and copper at pH 7.0. The Fe(III)-epinephrine complex was much more stable than was the Cu(II)-epinephrine complex. Oxygen consumption studies of ceruloplasmin catalyzed epinephrine oxidation showed that copper was a better promoter of epinephrine oxidation than was iron, suggesting that ceruloplasmin-catalyzed epinephrine oxidation results from adventitious copper bound to the purified enzyme. In light of these results, the physiological relevance of ceruloplasmin catalyzed oxidation of biogenic amines may be minor.     

Heterologous expression, purification, and characterization of recombinant rat cysteine dioxygenase

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K(m) value of 2.5 +/- 0.4 mm at pH 7.5 and 37 degrees C. The enzyme was shown to be specific for l-cysteine oxidation, whereas homocysteine inhibited CDO activity.     

A new prolyl hydroxylase acting on poly-L-proline, from suspension cultured cells of Vinca rosea

A new prolyl hydroxylase having a novel substrate specificity was isolated from the suspension-cultured cells of Vinca rosea. This enzyme was solubilized with 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1% Triton X-100, 0.3 M NaCl and 0.5 mM beta-mercaptoethanol from the membrane fractions of the cells, and was partially purified by (NH4)2SO4 fractionation and DEAE-Sephadex A-50 column chromatography. The enzyme preparation was found to require O2, Fe2+, ascorbate, alpha-ketoglutarate and poly-L-proline to attain maximum activity. The plant enzyme does not hydroxylate free proline and di-, tri- and tetra-L-proline, but hydroxylates octa-L-proline and poly-L-proline (Mr greater than 2000). Model peptides of unhydroxylated collagen, (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 are poor substrates for the plant enzyme. This means that the plant enzyme has a novel substrate specificity in regard to peptidyl substrate, and this differs from vertebrate prolyl hydroxylase, proline,2-oxoglutarate dioxygenase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase, EC 1.14.11.2).     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

4-Hydroxyphenylpyruvate dioxygenase is an iron-tyrosinate protein

A resonance Raman investigation into the blue chromophore of 4-hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron-tyrosinate proteins.     

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase from pseudomonas sp. strain P.J. 874

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 were studied with 14C- and different 3H-labelled 4-hydroxyphenylpyruvate. Tritium of ring-2,6-3H2-labelled substrate was released into water in 1:2 stoichiometry to 14CO2 formation. The tritium release from ring-3,5-3H2- and side chain-3-3H1-labelled 4-hydroxyphenylpyruvate was low as compared with 14CO2 formation. The apparent tritium isotope effects were below two, as judged by comparison of 3H/14C ratios of 4-hydroxyphenylpyruvate and homogentisate. The ratios showed no dependence on oxygen concentrations between 1 and 21% in the gas phase. Thus, a tritium assay can be used to determine the activity of 4-hydroxyphenylpyruvate dioxygenase. Apparently, none of the substrate hydrogens is involved in any rate-limiting step up to the first irreversible step. enol-4-Hydroxyphenylpyruvate was excluded as the active substrate tautomer.     

Characterization of 2,4-Dinitrotoluene Dioxygenase from Recombinant Esherichia coli Strain PFJS39: Its Direct Interaction with Vitreoscilla Hemoglobin

Burkholderia dinitrotoluene (DNT) dioxygenase in this study (from recombinant Esherichia coli strain PFJS39) is probably a multicomponent enzyme system that oxidizes 2,4-dinitrotoluene (DNT) to 4-methyl-5-nitrocatechol (MNC). DNT dioxygenase was purified by a four-step procedure that utilized consecutive gel filtration chromatography and a nondenaturing gel system. The purified enzyme oxidized DNT only in the presence of NADH and its yield increased by lipase pretreatment of crude cytosol. An estimated molecular weight of 100,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three subunits for the samples from consecutive gel filtration chromatography and nondenaturing PAGE. Their molecular weights were 52,000–71,000, 23,000–25,500, and 12,000–16,500. These results suggest that DNT dioxygenase exists as a heterotrimer. The K _M of DNT dioxygenase for O₂ is 50 μ M, consistent with inhibition results of DNT dioxygenase by Vitreoscilla hemoglobin (its K _M for O₂ is 7 μ M). The K _M for DNT is 180 μ M. The purified enzyme is relatively stable below 40°C, retains activity over a broad pH range, and is stimulated by several cofactors in addition to NADH.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Purification and Some Properties of Lignostilbene-a,/i-dioxygenase Responsible for the Ca—Cp Cleavage of a Diarylpropane Type Lignin Model Compound from Pseudomonas sp. TMY1009

A novel dioxygenase, lignostilbene-a,β-dioxygenase (LSD), which catalyzes cleavage of the interphenyl double bond of lignin-derived stilbenes, was isolated. Four isozymes of LSD were separated from cell-free extracts of Pseudomonas sp. TMY1009 by ion-exchange chromatography on a DEAE- Toyopearl column. The major isozyme, LSD-I, was purified to electrophoretic homogeneity and characterized.

LSD-I cleaved the interphenyl double bond of l,2-bis(4′-hydroxy-3′-methoxyphenyl)ethylene with the optimum pH at 8.5. The Km of LSD-I was 11 μm for the stilbene and 110/iM for oxygen. The molecular weight of LSD-I, which is composed of two identical subunits, was estimated to be 94,000. LSD-I contained 1 g atom of iron per 1 mol of enzyme protein.     

Purification and characterization of levoglucosan kinase from <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis> YZ-215

A protein named as levoglucosan kinase (EC 2.7.-.-)was purified to homogeneity from a wild isolated strain of Lipomyces starkeyi YZ-215. The protein was purified approximately 30-fold by conventional ammonium sulphate fractionation followed by Resource Q chromatography and two steps of Superdex 200 chromatography, and its physical and kinetic properties were investigated. The purified enzyme showed a molecular weight of 48 kDa by SDS-PAGE and 47.7 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), respectively. The enzyme was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0. Kinetic constants (apparent K _m values) for levoglucosan and ATP were 68.6 ± 13.7 mM and 0.68 ± 0.06 mM, respectively. After in-gel digestion by trypsin, three peptides were sequenced and analyzed by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Data of the amino acid sequences indicated that this protein might be a novel kinase. The purification of levoglucosan kinase from L. starkeyi YZ-215 represented a fundamental step to provide insights into the efficient utilization of cellulosic pyrolysate by bioconversion.     

Chitin Coated Cellulose as an Adsorbent of Lysozyme-like Enzymes: Some Applications

Galactose oxidase was purified from the culture supernatant of Gibberella fujikuroi by ammonium sulfate precipitation, chromatographies on DEAE-cellulose and hydroxylapatite, and gel filtration on Bio-Gel P-100. The purified enzyme had a molecular weight of 90,000 and an isoelectric point of pH 3.7, and contained about one atom of copper and about one atom of iron per mol of the enzyme protein. The enzyme was markedly inactivated by a copper-chelating agent, diethyldithiocarbamate, and reducing agents. The apoenzyme preparing on treatment of the enzyme with diethyldithiocarbamate could be reactivated only by the addition of either Cu⁺ or Cu^{2 +}. These results indicate that copper is involved in galactose oxidase activity of G. fujikuroi.     

Characterization of cytochrome oxidase purified from rat liver

The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc ₁. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS ^20,w of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000.     

Biochemical Properties of a Cephalosporin β-Lactamase from Pseudomonas aeruginosa

The cephalosporin β-lactamase from Pseudomonas aeruginosa GN918 was purified using CM-Sepha-dex column chromatography. The resulting preparation gave a single protein peak on electrofocusing column chromatography and a single protein band on polyacrylamide-gel electrophoresis. The specific enzyme activity was 22 950 units per mg of the purified enzyme protein. The optimal pH was 7.5 and the optimal temperature was 40 C for the hydrolysis of cephaloridine. Isoelectric point was 8.7 and the approximate molecular weight of the enzyme was found to be 34 000±2000. The enzyme activity was inhibited by iodine, p-chloromercuribenzoate and semi-synthetic penicillins. The enzymological properties of the isolated preparation have been compared with β-lactamases derived from other gram-negative enteric bacteria.     

Structural and functional characterization of 4-hydroxyphenylpyruvate dioxygenase from the thermoacidophilic archaeon Picrophilus torridus

4-Hydroxyphenylpyruvate dioxygenase (Hpd, EC 1.13.11.27) catalyzes the conversion of 4-hydroxyphenylpyruvate into homogentisate in the second step of oxidative tyrosine catabolism. This pathway is known from bacteria and eukaryotes, but so far no archaeal Hpd has been described. Here, we report the biochemical characterization of an Hpd from the extremophilic archaeon Picrophilus torridus (Pt_Hpd), together with its three-dimensional structure at a resolution of 2.6 Å. Two pH optima were observed at 50 °C: pH 4.0 (close to native conditions) and pH 7.0. The enzyme showed only moderate thermostability and was inactivated with a half-life of ~1.5 h even under optimal reaction conditions. At the ideal physiological growth conditions of P. torridus, Pt_Hpd was inactive after 1 h, showing that the enzyme is protected in vivo from denaturation and/or is only partially adapted to the harsh environmental conditions in the cytosol of P. torridus. The influence of different additives on the activity was investigated. Pt_Hpd exhibited a turnover number k _cat of 9.9 ± 0.6 s^?1 and a substrate binding affinity K _m of 142 ± 23 µM. In addition, substrate inhibition with a binding affinity K _i of 1.9 ± 0.3 mM was observed. Pt_Hpd is compared with isoenzymes from other species and the putative bacterial origin of the gene is discussed.     

The molecular properties of the copper enzyme galactose oxidase

Galactose oxidase (EC 1.1.3.9) has been purified 140-fold by DEAE- and CM-cellulose chromatography from cultures of Polyporus circinatus. The enzyme has a molecular weight of 68,000 ± 3,000 as determined by sedimentation equilibrium, sodium dodecyl sulfate-acrylamide gel electrophoresis, Sephadex G-150 chromatography, and osmometry. Galactose oxidase is a single-chain protein which does not self-associate. Charge isozymes of the enzyme are detected by ion-exchange chromatography and gel electrophoresis. The amino acid composition determined herein is significantly different from that previously reported (Kelly-Falcoz, F., Greenberg, H., And Horecker, B. L. (1965) J. Biol. Chem.240, 2966–2970). The enzyme contains 1% by weight of neutral carbohydrate.Galactose oxidase contains 1 g-atom of copper per 70,000 g of protein. The metal does not contribute to the electrophoretic or isozymic properties of the protein. However, the sedimentation coefficients of the holo- and apoenzymes, 4.76S and 4.83S, respectively, do suggest that small differences in protein conformation accompany the removal of the copper from the holoenzyme.Attempted sulfhydryl group titration of galactose oxidase shows that the holoenzyme is resistant to denaturation. However, in β-mercaptoethanol-guanidine HCl 5 half-cystine residues are titrated in the apoenzyme. On a dry-weight basis, the E_1cm^1% value for galactose oxidase at 280 nm is 15.4. Galactose oxidase has an isoelectric point above pH 10 which is a probable source of some of its anomalous behavior in physical measurements and enzyme-activity determinations.     

Dioxygenase and Co-oxidase activities of rat hepatic cytosolic lipoxygenase

The role of rat liver cytosolic lipoxygenase in the metabolism of benzidine was studied using linoleic acid as a cosubstrate. Under optimum assay conditions, cytosolic dioxygenase activity in the presence of 3.5 mM linoleic acid at pH 7.2 was 74.07 ± 1.43 nmoles/min/mg protein. Benzidine was oxidized at the rate of 3.18 ± 0.13 nmoles/min/mg cytosolic protein to benzidine diimine at pH 7.2 in the presence of 3.65 mM linoleic acid. Both dioxygenase and cooxidase reactions were inhibited by nordihydroguaiaretic acid in a concentration-dependent manner. Partially purified preparations of rat liver lipoxygenase, free of hemoglobin, exhibited a dioxygenase activity of 223.1 ± 65.9 nmoles/min/mg protein and cooxidase activity of 6.1 ± 0.5 nmoles/min/mg protein toward benzidine. These results suggest that hepatic lipoxygenase may play an important role in the metabolism of this hepatocarcinogen.     

Anthranilate hydroxylase from Aspergillus niger: new type of NADPH-linked nonheme iron monooxygenase

Anthranilate hydroxylase from Aspergillus niger catalyzes the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid. This enzyme has been purified to homogeneity and has a molecular weight of 89,000. The enzyme is composed of two subunits of 42,000 with 2 gram-atoms of nonheme iron per mol. Fe2+-chelators like alpha,alpha'-dipyridyl and o-phenanthroline are potent inhibitors of the enzyme activity. Absorption and fluorescence spectra of the enzyme offer no evidence for the presence of other cofactors like flavin. Flavins and flavin-specific inhibitors like atebrin have no effect on the activity of the enzyme. The enzyme incorporates one atom of oxygen each from 18O2 and H218O into the product 2,3-dihydroxybenzoic acid. Based on these studies, it is concluded that anthranilate hydroxylase from A. niger is a new type of NADPH-linked nonheme iron monooxygenase.     

Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.

Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field.