- The structure of 4-hydroxylphenylpyruvate dioxygenase complexed with 4-hydroxylphenylpyruvic acid reveals an unexpected inhibition mechanism
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4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for both drug and pesticide discovery. As a typical Fe(II)-dependent dioxygenase, HPPD catalyzes the complicated transformation of 4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid (HGA). The binding mode of HPPA in the catalytic pocket of HPPD is a focus of research interests. Recently, we reported the crystal structure of Arabidopsis thaliana HPPD (AtHPPD) complexed with HPPA and a cobalt ion, which was supposed to mimic the pre-reactive structure of AtHPPD-HPPA-Fe(II). Unexpectedly, the present study shows that the restored AtHPPD-HPPA-Fe(II) complex is still nonreactive toward the bound dioxygen. QM/MM and QM calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of its phenyl ring, and the residue Phe381 plays a key role in orienting the phenyl ring. Kinetic study on the F381A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an intermediate of the substrate transportation instead of the pre-reactive complex. More importantly, the binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors, suggesting that these inhibitors resist the association of dioxygen (and exert their inhibitory roles) in the same way as the HPPA. The present study provides insights into the inhibition mechanism of HPPD inhibitors.
- Wang, Xiaoning,Lin, Hongyan,Liu, Junjun,Zhao, Xinyun,Chen, Xi,Yang, Wenchao,Yang, Guangfu,Zhan, Chang-guo
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- Initial studies on the substrate specificity of soluble recombinant 4- hydroxyphenylpyruvate dioxygenase from rat liver
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The molecular cloning and expression of the cDNA encoding 4- hydroxyphenylpyruvate dioxygenase (rat F antigen) into E. coli and initial studies concerning the substrate specificity of the recombinant protein are described.
- Crouch,Baldwin,Lee,MacKinnon,Zhang
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- Toward a high added value compound 3, 4-dihydroxyphenylacetic acid by electrochemical conversion of phenylacetic acid
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Abstract The development of the effective procedure to recover the potentially high-added-value phenolic compound, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) was investigated using electrochemical conversion of phenylacetic acid (PAA). The proposed mechanism is based on the hypothesis of two-electron oxidation of PAA molecule leading to 3-hydroxyphenyl acetic acid. The latter underwent a second bi-electronic transfer by means of a radical cation, thus leading to the formation of the 2,5 dihydroxyphenylacetic (2,5-DHPAA) acid and 3,4-DHPAA as major products. The 3,4-DHPAA was synthesized by anodic oxidation of PAA at lead dioxide electrode and identified by cyclic voltammetry and spectrophotometry UV-visible. It was also confirmed by mass spectrophotometry using LC-MS/MS apparatus. According to their voltammetric behavior during electrolysis, the oxidation potential of 3,4-DHPAA was lower than that of PAA. The antioxidant activity was measured by DPPH assay, showing that the strongest antiradical activity was detected when the 3,4-DHPAA concentration was higher during electrolysis experiments.
- Trabelsi, Souhel Kallel,Dridi Gargouri, Olfa,Gargouri, Boutheina,Abdelhèdi, Ridha,Bouaziz, Mohamed
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p. 370 - 376
(2015/05/27)
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- Phytotoxic effects of selected N-benzyl-benzoylhydroxamic acid metallo-oxygenase inhibitors: Investigation into mechanism of action
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Treatment of Arabidopsis thaliana with 100 μM hydroxamic acids F1 and F2, found previously to inhibit carotenoid cleavage dioxygenase enzyme CCD1, was found to cause chlorophyll bleaching and phytotoxicity. A further set of hydroxamic acid analogues was synthesised, and these compounds were found to be phytotoxic towards A. thaliana at 16-400 μM, and to show some phytoxicity towards broad-leaved weeds C. album and S. media at 100 μM. Compound F1 was found to inhibit p-hydroxy-phenylpyruvate dioxygenase (HPPD), a known herbicide target (IC50 30 μM), but compounds F5 and F8 showed no inhibition of HPPD, despite F8 showing higher levels of phytotoxicity. Plants grown in the presence of F1 or F5 that were treated with 50 μM homogentisic acid showed partial recovery of growth, indicating some inhibition of HPPD in planta. These are the first hydroxamic acid inhibitors reported for HPPD, but the results indicate that inhibition of HPPD is only partly responsible for the observed phytotoxicity, and that another unknown metalloenzyme is also targeted by these compounds.
- Sergeant, Martin J.,Harrison, Peter J.,Jenkins, Robert,Moran, Graham R.,Bugg, Timothy D. H.,Thompson, Andrew J.
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p. 3461 - 3465
(2013/11/06)
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- Intermediate partitioning kinetic isotope effects for the NIH shift of 4-hydroxyphenylpyruvate dioxygenase and the hydroxylation reaction of hydroxymandelate synthase reveal mechanistic complexity
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4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3′-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3′-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3′-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp3 hybridization in a HPPD variant and sp 2 hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.
- Shah, Dhara D.,Conrad, John A.,Moran, Graham R.
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p. 6097 - 6107
(2013/09/23)
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- Herbicide-tolerant plants through bypassing metabolic pathway
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The invention concerns a novel method for making herbicide-tolerant plants, in particular to HPPD inhibiting herbicides, the nucleic acid sequences coding for enzymes capable of being used in said method, expression cassettes containing them and transgenic plants comprising at least one of said expression cassettes.
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- Development of a Novel Biocatalyst for the Resolution of rac-Pantolactone
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A novel L-pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L-pantolactone to L-pantoic acid yielding D-pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa-Zn2+-hydrolase with a Km for L-pantolactone of 7 mM and a Vmax of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of Ptac and Prha yielding enzyme activities of up to 600 U/g dry weight. Resolution of D,L-pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D-pantolactone with 50% yield and 90-95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D-pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.
- Kesseler, Maria,Friedrich, Thomas,Hoeffken, Hans Wolfgang,Hauer, Bernhard
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p. 1103 - 1110
(2007/10/03)
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- Hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection
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Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydropyranyl etherp-nitrophenyl ester) and HLTE (homogentisic γ-lactone tetrahydropyranyl ether), are described for the chemical derivatization of primary and/or secondary amines to form an electrochemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible electrochemical detection at mild oxidation potentials. The reactivity of each reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipeptide isoleucine leucine methyl ester as model analytes. The investigation included the isolation and identification of the intermediates and final products from derivatization of EBzA. These isolated standards were subsequently characterized with respect to electrochemical properties by means of cyclic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product was determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl-] = 3 M). The CLOD values obtained by LC-EC after derivatization of aqueous solutions of EBzA and Ile-Leu-OMe with NDTE were 25 nM (250 fmol) and 250 nM (2.5 pmol), respectively.
- Rose, Mark J.
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p. 2221 - 2230
(2007/10/03)
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- Copolyesters, molded articles from the same and aromatic triols used for producing the same
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A copolyester consisting essentially of ethylene terephthalate units and further comprising 0.01 to 1 mole % based on the sum of the moles of total diol units and the moles of triol units of a triol unit represented by the following formula (I) and/or a triol unit represented by the following formula (II). STR1 wherein A is a group represented by formula --CH2 CH2 -- or formula --CH(CH3)CH2 --, B is a divalent hydrocarbon group, --CO--, --SO2 --, --0-- or a direct bond (-), and p, q, r, s, t and u are each an integer of 1 to 8. A process for producing the same and molded articles (in particular extrusion blow molded articles) therefrom. The copolyesters of the present invention are applicable to melt moldings accompanying melt extrusion, in particular extrusion blow molding, where parisons therefrom have good draw-down tendency and blow moldability. The copolyesters can give, without causing troubles on molding, molded articles, in particular extrusion blow molded articles, having excellent transparency, heat resistance, moisture resistance and like properties. Aromatic triols usable for producing the above copolyesters are also provided.
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- Synthesis of homogentisic acid by carbonylation
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The synthesis of homogentisic acid by carbonylation of 2,5-dihydroxybenzyl chloride derivative followed by hydrolysis is discussed.
- Prasad, Chalasani S N,Adapa, Srinivas R
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p. 626 - 627
(2007/10/02)
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- PHASEOLOIDIN, A HOMOGENTISIC ACID GLUCOSIDE FROM ENTADA PHASEOLOIDES
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The structure of phaseoloidine isolated from the seeds of Entada phaseolides has been determined as homogentisic acid 2-O-β-D-glucopyranoside by chemical and spectral means.
- Barua,, Arun K.,Chakrabarty, Manas,Datta, Pran K.,Ray, Sarmila
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p. 3259 - 3262
(2007/10/02)
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- One-Electron Redox Potentials of Phenols. Hydroxy- and Aminophenols and Related Compounds of Biological Interest
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The rate constants for reversible electron transfer between a series of substituted phenolate ions and anilines and various substituted phenoxyl or anilino radicals in aqueous solution were measured by observing the formation or depletion of the radicals involved.Nonequilibrium concentrations of the radicals were produced in the presence of the corresponding phenols or anilines by using the pulse radiolysis technique.The relaxation of the system to equilibrium was monitored by optical detection methods.From the equilibrium constants for one-electron transfer, the one-electron redox potentials (E2) for 38 phenolic or anilino type compounds were determined, many of which are natural products.The redox potentials are strongly influenced by electron-donating or -withdrawing substituents at the aromatic system.
- Steenken, S.,Neta, P.
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p. 3661 - 3667
(2007/10/02)
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