A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase

Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2′,4′,6′,4–tetrahydroxychalcone). Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase. Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time. Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o‐hydroxylation of the monophenol THC to the o‐diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the corresponding o‐quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K_m = 0.12 mM, V_m = 13 μM/min, V_m/K_m = 0.11/min. Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone. Copyright © 2009 John Wiley & Sons, Ltd.     

Homology modeling and dynamics study of aureusidin synthase--an important enzyme in aurone biosynthesis of snapdragon flower

Aurones, a class of plant flavonoids, provide bright yellow color on some important ornamental flowers, such as cosmos, coreopsis, and snapdragon (Antirrhinum majus). Recently, it has been elucidated that aureusidin synthase (AUS), a homolog of plant polyphenol oxidase (PPO), plays a key role in the yellow coloration of snapdragon flowers. In addition, it has been shown that AUS is a chalcone-specific PPO specialized for aurone biosynthesis. AUS gene has been successfully demonstrated as an attractive tool to engineer yellow flowers in blue flowers. Despite these biological studies, the structural basis for the specificity of substrate interactions of AUS remains elusive. In this study, we performed homology modeling of AUS using Grenache PPO and Sweet potato catechol oxidase (CO). An AUS-inhibitor was then developed from the initial homology model based on the CO and subsequently validated. We performed a thorough study between AUS and PTU inhibitor by means of interaction energy, which indicated the most important residues in the active site that are highly conserved. Analysis of the molecular dynamics simulations of the apo enzyme and ligand-bound complex showed that complex is relatively stable than apo and the active sites of both systems are flexible. The results from this study provide very helpful information to understand the structure-function relationships of AUS.     

On the role of superoxide in reactions catalyzed by rubredoxin of Pseudomonas oleovorans

In the presence of NADH, and the reductase and rubredoxin components of the ω-hydroxylation system of $\text{Pseudomonas oleovorans}$, epinephrine is oxidized to adrenochrome at pH 7.8, and the reaction is strongly inhibited by the addition of superoxide dismutase (SDM). Boiled SDM has no effect on the reaction rate. The oxidation reaction is oxygen-dependent, and approximately 1 mole of H₂O₂ is produced per mole of O₂ consumed. The stoichiometry between NADH oxidation and adrenochrome formation is approximately 2:1. Epoxidation and epinephrine oxidation are mutually competitive reactions, despite the fact that the epoxidation reaction is not stimulated by a superoxide generating system nor inhibited by SDM.     

Altered leaf colour is associated with increased superoxide‐scavenging activity in aureusidin‐producing transgenic plants

The health‐promoting property of diets rich in fruits and vegetables is based, in part, on the additive and synergistic effects of multiple antioxidants. In an attempt to further enhance food quality, we introduced into crops the capability to synthesize a yellow antioxidant, aureusidin, that is normally produced only by some ornamental plants. For this purpose, the snapdragon (Antirrhinum majus) chalcone 4′‐O‐glucosyltransferase (Am4’CGT) and aureusidin synthase (AmAs1) genes, which catalyse the synthesis of aureusidin from chalcone, were expressed in tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa) plants that displayed a functionally active chalcone/flavanone biosynthetic pathway. Leaves of the resulting transgenic plants developed a yellow hue and displayed higher superoxide dismutase (SOD) inhibiting and oxygen radical absorbance capacity (ORAC) activities than control leaves. Our results suggest that the nutritional qualities of leafy vegetables can be enhanced through the introduction of aurone biosynthetic pathways.     

Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.     

Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H 4 and -2′,3′,4′,5′,5″- 2H 5

Abstract

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H ₄ and -2′,3′,4′,5′,5″- ²H ₅ from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-α,β-D-allofuranose-3,4-d ₂ 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 16 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ) or to 1-O-methyl-3,5-di-O-benzyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 18 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ).     

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H₂O₂ and Br^?) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H₂O₂ or Br^?) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H₂O₂ + Br^?) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane-or lipoprotein-associated unsaturated lipids in centers of inflammation.     

What colour of flowers do Lepidoptera prefer for foraging?

Food plant preferences of some Lepidoptera species associated with particular colour of the flowers were investigated. Based on 1,329 field observations of 43 Lepidoptera and 66 plant species, Lepidoptera showed a high tendency (G-test, G _adj = 698.6, df = 6, P < 0.001) to use the yellow (29%) and pink (28%) coloured flowers for foraging. Compared to the other colours it was evident that plants with red flowers (2%) were not preferred. Moreover, the plants with red (H = 0.435) and yellow-white (H = 0.543) flowers were not visited by diverse Lepidoptera species. Although yellow and pink flowers were most frequently visited, the highest degrees of the Lepidoptera diversity values were associated with the plants having blue (H = 0.647) and purple (H = 0.634) flowers. Species of Nymphalidae were most numerous (14 spp.) in the study area and the members of this family were observed 430 times on 39 different plant species, but never on plants with red flowers. Pieris rapae was the most abundant species that occurred 136 times on a total of 21 different plant species of which eight had yellow flowers. But, this species has never been seen while feeding on red flowers.     

Regulation of the activity of glyceraldehyde 3-phosphate dehydrogenase by glutathione and H2O2

The activity lost during storage of a solution of muscle glyceraldehyde 3-phosphate dehydrogenase was rapidly restored on adding a thiol compound, but not arsenite or azide. On treatment with H₂O₂, the enzyme was partially inactivated and complete loss of activity occurred in the presence of glutathione. Samples of the enzyme pretreated with glutathione followed by removal of the thiol compound by filtration on a Sephadex column showed both full activity and its complete loss on adding H₂O₂, in the absence of added glutathione. Most of the activity was restored when the H₂O₂-inactivated enzyme was incubated with glutathione (25mM) or dithiothreitol (5mM) whereas arsenite or azide were partly effective and ascorbate was ineffective. The need for incubation for a long time with a strong reducing agent for restoration of activity suggests that the oxidized group (disulfide or sulfenate) must be in a masked state in the H₂O₂-inactivated enzyme. Analysis by SDS-PAGE gave evidence for the formation of a small quantity of glutathione-reversible disulfide-form of the enzyme. Circular dichroic spectra indicated a decrease in -helical content in the inactivated form of the enzyme. The evidence suggest that glutathione and H₂O₂ can regulate the active state of this enzyme.     

Effect of Modified Fenton’s Reaction on Microbial Activity and Removal of PAHs in Creosote Oil Contaminated Soil

This study describes the removal of polycyclic aromatic hydrocarbons (PAHs) from creosote oil contaminated soil by modified Fenton’s reaction in laboratory-scale column experiments and subsequent aerobic biodegradation of PAHs by indigenous bacteria during incubation of the soil. The effect of hydrogen peroxide addition for 4 and 10 days and saturation of soil with H₂O₂ on was studied. In both experiments the H₂O₂ dosage was 0.4 g H₂O₂/g soil. In completely H₂O₂−saturated soil the removal of PAHs (44% within 4 days) by modified Fenton reaction was uniform over the entire soil column. In non-uniformly saturated soil, PAH removal was higher in completely saturated soil (52% in 10 days) compared to partially saturated soil, with only 25% in 10 days. The effect of the modified Fenton’s reaction on the microbial activity in the soil was assessed based on toxicity tests towards Vibrio fischeri, enumeration of viable and dead cells, microbial extracellular enzyme activity, and oxygen consumption and carbon dioxide production during soil incubation. During the laboratory-scale column experiments, the toxicity of column leachate towards Vibrio fischeri increased as a result of the modified Fenton’s reaction. The activities of the microbial extracellular enzymes acetate- and acidic phosphomono-esterase were lower in the incubated modified Fenton’s treated soil compared to extracellular enzyme activities in untreated soil. Abundance of viable cells was lower in incubated modified Fenton treated soil than in untreated soil. Incubation of soil in serum bottles at 20 °C resulted in consumption of oxygen and formation of carbon dioxide, indicating aerobic biodegradation of organic compounds. In untreated soil 20–30% of the PAHs were biodegraded during 2 months of incubation. Incubation of chemically treated soil slightly increased PAH-removal compared to PAH-removal in untreated soil.     

Localization of partially methylated flavonol glucosides in Chrysosplenium americanum. I. Preparation and some properties of a trimethyl flavonol glucoside antibody

5,2′,5′-Trihydroxy-3,7,4′-trimethoxyflavone-2′-O-glucoside, a major flavonoid constituent of Chrysosplenium americanum Schwein. ex Hooker was conjugated to bovine serum albumin (BSA) by the diazo reaction in good yield and with a molar ratio of 11.5:1. Antibody raised against the latter conjugate had a titer value of 1:1600 and was found to be specific for the 2′-O-glucosides of tri- and tetramethoxyflavones. Some cross reactivity (about 55%) was observed against the pentamethoxyflavone-5′-O-glucoside; but almost none with the parent hydroxyflavone, quercetin, or any of its partially methylated (3,7,4′-tri- or 3,7,3′,4′-tetramethyl-) derivatives. The specificity of antibodies raised against the 2′-O-glucosides of Chrysosplenium makes them useful for the intracellular localization of these natural constituents.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

On the rate-limiting step in W-hydroxylation of lauric acid

Incubation of lauric acid with rat liver microsomes and NADPH yielded a mixture of 11-D-, 11-L-, and 12-hydroxylauric acids. 11-L- and 11-D-hydroxylations of 11-²H₂-lauric acid were accompanied by marked isotope effects, indicating that in these reactions splitting of the CH bond is rate limiting. When 11- and 12-hydroxylations of lauric acid were carried out in D₂O, 12-hydroxylation but not 11-hydroxylation was inhibited, showing that different steps are rate limiting in the two reactions.     

Involvement of copper amine oxidase (CuAO)-dependent hydrogen peroxide synthesis in ethylene-induced stomatal closure in Vicia faba

Ethylene promotes stomatal closure via inducing hydrogen peroxide (H₂O₂) generation. H₂O₂ can be catalytically synthesized by several enzymes in plants. Here, by means of stomatal bioassay, the analysis of enzyme activity and using laser-scanning confocal microscopy based on the H₂O₂-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), the roles of copper amine oxidase (CuAO) in ethylene-induced H₂O₂ production in guard cells and stomatal closure in Vicia faba L. were investigated. 1-aminocyclopropane-1-carboxylic acid (ACC), an immediate precursor of ethylene synthesis, and ethylene gas significantly activated CuAO in intercellular washing fluid from leaves, the production of H₂O₂ in guard cells, and stomatal closure. These effects of ACC and ethylene gas were largely prevented by both aminoguanidine and 2-bromoethylamine, which are irreversible inhibitors of CuAO. Among major catalyzed and metabolized products of CuAO, only H₂O₂ could markedly promote stomatal closure and evidently reversed the effect of CuAO inhibitor on stomatal closure by ACC and ethylene gas. The data described above show that CuAO-mediated H₂O₂ production is involved in ethylene-induced stomatal closure.     

The reaction of copper-2,9-dimethyl-1,10-phenanthroline with hydrogen peroxide

The copper complex of 2,9-dimethyl-1,10-phenanthroline(2,9-dmp) is accumulated by a variety of organisms and is highly toxic. Bioaccumulation depends on reduction of copper(II) to (I), since only the copper(I)-2,9-dmp complex is lipophilic. In the case of the marine diatom, Nitzschia closterium, it is proposed that hydrogen peroxide, produced by the algae during photosynthesis, is the in vivo reductant. Hydrogen peroxide rapidly reduces copper(II)-2,9-dmp, but an excess of H₂O₂ leads to destruction of the yellow copper(I) complex. Rate constants for the formation and degradation of the yellow complex are reported. Oxygen, light, and a hydroxylating agent are released during the degradation reaction. A reaction mechanism is proposed for the destruction of copper-2,9-dmp by excess H₂O₂, involving attack on the 5, 6 positions of the phenanthroline ring by hydroxyl radical, then further oxidation by singlet oxygen and H₂O₂. These in vivo degradation reactions are believed to be the cause of the extreme toxicity of the complex.     

Acyl-CoA oxidase of rat liver: A new enzyme for fatty acid oxidation

Acyl-CoA oxidase was purified from rat liver based on the palmitoyl-CoA-dependent H₂O₂-forming activity. Enoyl-CoA formation from palmitoyl-CoA by this enzyme was shown by the following observations; first, palmitoyl-CoA-dependent NAD⁺ reduction in the presence of this enzyme, crotonase, and 3-hydroxyacyl-CoA dehydrogenase, and, second, palmitoyl-CoA-dependent increase in absorbance at 263 nm. Same amounts of enoyl-CoA and H₂O₂ were formed during the reaction. It is concluded that this enzyme catalyzes the following reaction: Palmitoyl-CoA + O₂ → $\text{transm}$-2-Hexadecenoyl-CoA + H₂O₂. It was most active toward C₁₂-C₁₈ acyl-CoAs. C₂₀ and C₂₂ acyl-CoAs were also oxidized, but C₄ and C₆ acyl-CoAs were hardly oxidized at all.     

Possible Mechanism of Oxygen Activation in Linoleate Peroxidation by Fusarium Lipoxygenase

The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxygenase, was studied. The enzyme did not require any cofactors, such as H₂O₂, however, both superoxide dismutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H₂O₂ caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O₂? and H₂O₂ are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase. This peroxidation reaction was also prevented by scavengers of singlet oxygen (¹O₂), but not by scavengers of hydroxy 1 radical (OH). Generation of O₂? in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide formation was greater in the D₂O than in the H₂O buffer system. These results suggest that the Haber–Weiss reaction (O₂^?+H₂O₂→OH^?+OH^·+¹O₂) is taking part in linoleate peroxidation by Fusarium lipoxygenase, and the ¹O₂ evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other heme-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.     

Chalcone glycosides of Antirrhinum majus

Three chalcones have been found in yellow flowers of A. majus, two of which have been identified as chalcononaringenin 4′-glucoside and 3,4,2′,4′,6′-pentahydroxychalcone 4′-glucoside.     

The novel non-heme vanadium bromoperoxidase from marine algae: Phosphate inactivation

Summary Vanadium bromoperoxidase is a naturally occurring vanadium-containing enzyme isolated from marine algae. V-BrPO catalyzes the oxidation of halides by hydrogen peroxide which can result in the halogenation of organic substrates. Bromoperoxidase activity is measured by the halogenation of monochlorodimedone (2-chloro-5,5-dimethyl-1,3-dimedone, MCD). In the absence of an organic substrate, V-BrPO catalyzes the halide-assisted disproportionation of hydrogen peroxide yielding dioxygen. The dioxygen formed is in the singlet excited state (¹O₂). V-BrPO is quite stable to thermal denaturation and denaturation by certain organic solvents which makes V-BrPO an excellent candidate for industrial applications. The stability of V-BrPO in the presence of strong oxidants and in the presence of phosphate is reported. Incubation of V-BrPO in phosphate buffer (1–100 mM at pH 6; 2–10 mM at pH 5) inactivates the enzyme. The inactivity can be fully restored by the addition of vanadate if excess phosphate is removed. The inactivation of V-BrPO by phosphate can be prevented by the presence of H₂O₂ (4–40 mM). We are currently investigating the mechanism of V-BrPO inactivation by phosphate. V-BrPO was not inactivated by HOCl (1 mM) nor H₂O₂. In addition V-BrPO was not inactivated under turnover conditions of 1 mM H₂O₂ with 0.1–1 M Cl^– at pH 5 nor 2 mM H₂O₂ with 0.1 M Br^–.     

Studies on the Mechanism of the Oxidation of Tea Leaf Catechins

In order to understand the mechanism of the enzymic oxidation of epigallocatechin, ethyl gallate was taken up as a model substrate on account of its close structural similarity to the w-trihydroxyphenyl group, which indeed is supposed to be attacked by oxidase, when epigallocatechin is oxidized enzymically. By the action of tea or apple oxidase at pH 5.5, it gave a characteristic polyphenolic substance, which was obtained in prisms, C₁₈H₁₈O₁₀·2H₂O, and proved to be diethyl 4,4′,5,5′,6,6′-hexahydroxydiphenate, as the product was converted into ellagic acid by hydrolysis of ester linkage and then lactone formation.