Peroxidase Catalysis Research Articles

Wood-mimetic skins prepared using horseradish peroxidase catalysis to induce surface wrinkling of chitosan film upon drying

We previously developed bio-based wrinkled surfaces induced by wood-mimetic skins upon drying in which microscopic wrinkles were fabricated on a chitosan (CS) film by immersing it in a phenolic acid solution, followed by horseradish peroxidase (HRP)-catalyzed surface reaction and drying. However, the detailed structure of the resulting wood-mimetic skins, including crosslinking mode and thickness, has not been clarified due to the difficulty of the analysis. Here, we prepare wrinkled films using ferulic acid (FE), vanillic acid (VA), and homovanillic acid (HO) and characterize their structures to clarify the unknown characteristics of wood-mimetic skin. Chemical and structural analyses of wood-mimetic skins prepared using VA and HO indicate that the crosslinking structure in the skin is composed of ionic bonds between CS and an oligophenolic residue generated by the HRP-catalyzed reaction on the CS surface. Moreover, the quantity of these ionic bonds is related to the skin hardness and wrinkle size. Finally, SEM and TOF-SIMS analyses indicate that the skin thickness is on the submicron order (<200nm).

Superoxide Dismutase Dysregulation Undermines Endogenous Antioxidant System and Promote Retinal Neurodegenerative Pathology

Oxidative stress is generally associated with unregulated relationship between free radical production and enzymes responsible for clearing them. Endogenous antioxidant system protects various tissues and cells rendered susceptible to damage by exposure to excessive oxidative milieu. Superoxide dismutase is one of the major enzymes in living systems to regulate endogenous reactive oxygen species (ROS) levels. It partitions highly reactive superoxide radicals into less damaging oxygen or hydrogen peroxide (H2O2). Glutathione peroxidase and catalase are some of the other enzymes which play significant roles in maintaining the equilibrium of free radicals and further decompose H2O2. Catalase mediates decomposition of hydrogen peroxide into O2 and H2O while glutathione peroxidase catalyses reduction of lipid hydroperoxides and hydrogen peroxide to H2O. In the eye, ROS has particularly been implicated in mediating damage to retinal cells and other ocular tissues in diseases such as ocular hypertension, age related macular degeneration (AMD) and various inflammatory disorders of the eye.

Platinum(II)-Oligonucleotide Coordination Based Aptasensor for Simple and Selective Detection of Platinum Compounds

Wide use of platinum-based chemotherapeutic regimens for the treatment for carcinoma calls for a simple and selective detection of platinum compound in biological samples. On the basis of the platinum(II)-base pair coordination, a novel type of aptameric platform for platinum detection has been introduced. This chemiluminescence (CL) aptasensor consists of a designed streptavidin (SA) aptamer sequence in which several base pairs were replaced by G-G mismatches. Only in the presence of platinum, coordination occurs between the platinum and G-G base pairs as opposed to the hydrogen-bonded G-C base pairs, which leads to SA aptamer sequence activation, resulting in their binding to SA coated magnetic beads. These Pt-DNA coordination events were monitored by a simple and direct luminol-peroxide CL reaction through horseradish peroxidase (HRP) catalysis with a strong chemiluminescence emission. The validated ranges of quantification were 0.12-240 μM with a limit of detection of 60 nM and selectivity over other metal ions. This assay was also successfully used in urine sample determination. It will be a promising candidate for the detection of platinum in biomedical and environmental samples.

Biomimetic Nitration of Phenols Using Metalloporphyrins/H2O2/NO2 −

An efficient metalloporphyrins/H2O2/NO2 − nitration of phenols has been developed. The total yield of nitrophenol could reach up to 55.1 %, which is about 4 and sixfold higher than that of horse radish peroxidase catalysis and peroxynitrite nitration, respectively. Furthermore, the nitration system attained an enhanced regioselectivity of 1.0 o/p ratio, and exhibited a good substrate scope of monophenols. This protocol is environmentally friendly compared with HNO3/H2SO4 nitration, and stable, inexpensive and organic solvent tolerant compared with enzyme catalytic nitration and peroxynitrite nitration reaction.

Selenocysteine oxidation in glutathione peroxidase catalysis: an MS-supported quantum mechanics study

Glutathione peroxidases (GPxs) are enzymes working with either selenium or sulfur catalysis. They adopted diverse functions ranging from detoxification of H2O2 to redox signaling and differentiation. The relative stability of the selenoenzymes, however, remained enigmatic in view of the postulated involvement of a highly unstable selenenic acid form during catalysis. Nevertheless, density functional theory calculations obtained with a representative active site model verify the mechanistic concept of GPx catalysis and underscore its efficiency. However, they also allow that the selenenic acid, in the absence of the reducing substrate, reacts with a nitrogen in the active site. MS/MS analysis of oxidized rat GPx4 complies with the predicted structure, an 8-membered ring, in which selenium is bound as selenenylamide to the protein backbone. The intermediate can be re-integrated into the canonical GPx cycle by glutathione, whereas, under denaturing conditions, its selenium moiety undergoes β-cleavage with formation of a dehydro-alanine residue. The selenenylamide bypass prevents destruction of the redox center due to over-oxidation of the selenium or its elimination and likely allows fine-tuning of GPx activity or alternate substrate reactions for regulatory purposes.

Substrate cycling based fluorometric assay for dihydroxyacetone phosphate

A sensitive fluorometric assay for the determination of dihydroxyacetone phosphate (DHAP) is reported here. DHAP is reduced to l-glycerol-3-phosphate with NADH-dependent α-glycerophosphate dehydrogenase. DHAP recycling is provided by oxidation reaction catalysed by α-glycerophosphate oxidase to release hydrogen peroxide. The reaction of hydrogen peroxide with Amplex® Red reagent under horseradish peroxidase catalysis leads to the fluorescent product resorufin. The limit of detection of DHAP is estimated at 1pmol which is roughly 2250 fold more sensitive than the usual DHAP assay based on the detection of NADH by spectrophotometry. This assay is ready-to-use for automated medium-throughput screening.

Kinetics of Enzyme-Catalyzed Cross-Linking of Feruloylated Arabinan from Sugar Beet

Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.

Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes

Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues – via the generation of tyrosyl radicals (Tyr) – are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H2O2-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H2O2-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.

Mutagenesis Study on the Conformation of Distal Histidine in Dehaloperoxidase-Hemoglobin

Dehaloperoxidase-hemoglobin (DHP A), found in terebellid polychaete Amphitrite ornata, acts as a bi-functional protein. It has the globin fold and acts as an oxygen carrier, but it can also act as the peroxidase, which can oxidatively dehalogenate halophenols found in benthic ecosystems to the corresponding quinones. The distal histidine His55 is the only residue that determines the substrate and heme ligand binding function. His55 also plays a key role in peroxidase catalysis. His55 has two conformations, open conformation and closed conformation, which is related to inhibitor and substrate binding respectively. In myoglobin, Phe46 and Arg45 have been suggested the critical residues effect the C alpha - C beta rotation of distal His64. Moreover, in myoglobin the residue adjacent to His 64 (the distal histidine) is a glycine (Gly65). Although this is a conserved feature in many globins, DHP A has a threonine in this position. To understand the role of the amino acid in this position the mutants T56G, T56A, T56S and T56V were prepared. Kinetic assay results show that all of these mutants have higher activity than wild-type DHP A. Molecular dynamics simulations, Resonance Raman spectroscopy, X-ray crystallography and other methods will be presented to understand how a change in the Thr56 position affects the both the conformation of His55 and peroxidase activity. The inhibitor in DHP A binds in an unusual internal binding site. We will determine the inhibitor binding constants and measure the enzyme inhibition in kinetic assays to understand the role played by the distal histidine conformation inhibition as well.

Biodepollution of wastewater containing phenolic compounds from leather industry by plant peroxidases

This study deals with the use of peroxidases (POXs) from Allium sativum, Ipomoea batatas, Raphanus sativus and Sorghum bicolor to catalyze the degradation of free phenolic compounds as well as phenolic compounds contained in wastewater from leather industry. Secretory plant POXs were able to catalyze the oxidation of gallic acid, ferulic acid, 4-hydroxybenzoic acid, pyrogallol and 1,4-tyrosol prepared in ethanol 2% (v:v). Efficiency of peroxidase catalysis depends strongly on the chemical nature of phenolic substrates and on the botanical source of the enzymes. It appeared that POX from Raphanus sativus had the highest efficiency. Results show that POXs can also remove phenolic compounds present in industrial wastewater such as leather industry. Removal of phenolic compounds in wastewater from leather industry by POX was significantly enhanced by polyethylene glycol.

The effects of manganese and copper in vitro and in vivo on peroxidase catalytic cycles

Here we present the results of in vitro and in vivo studies of the influence of Mn 2+ and Cu 2+ on the peroxidative and oxidative catalytic functions of class III peroxidase. Complex peroxidase catalysis by intermediates generated in the reaction was analyzed by utilizing the activating effect of Mn 2+ and the inhibitory effect of Cu 2+ on the oxidative reaction in vitro. p-Coumaric acid was used as an enzyme substrate in the peroxidative reaction and as a cofactor in the oxidative reaction. In order to correlate the observed in vitro effects with the in vivo situation, we exposed maize plants to excess concentrations of Mn 2+ and Cu 2+ in the hydroponic solutions. Copper severely arrested plant growth, while manganese exerted no significant effect. The effects on peroxidase activity and isoforms profile of root soluble and cell wall bound fractions were studied. Inhibition of the peroxidase oxidative function by copper was reversible, localized in the cell wall, and accompanied by disappearance of some and appearance of new cationic isoforms. Copper-mediated changes were suppressed by the presence of manganese, although Mn 2+ treatment per se did not affect the activity of the peroxidase enzyme. The results on the peroxidase activity in maize roots grown with excess Mn 2+ and Cu 2+ point to the coupling between the oxidative cycle, root growth and different peroxidase isoforms.

On the Role of Water in Peroxidase Catalysis: A Theoretical Investigation of HRP Compound I Formation

We have investigated the dynamics of water molecules in the distal pocket of horseradish peroxidase to elucidate the role that they may play in the formation of the principal active species of the enzymatic cycle (compound I, Por(o+)-Fe(IV)=O) upon reaction of the resting Fe(III) state with hydrogen peroxide. The equilibrium molecular dynamics simulations show that, in accord with experimental evidence, the active site access channel is hydrated with an average of two to three water molecules within 5 A from the bound hydrogen peroxide. Although the channel is always hydrated, the specific conformations in which a water molecule bridges H(2)O(2) and the distal histidine, which were found (Derat; et al. J. Am. Chem. Soc. 2007, 129, 6346.) to display a low-energy barrier for the initial acid-base step of the reaction, occur with low probability but are relevant within the time scale of catalysis. Metadynamics simulations, which were used to reconstruct the free-energy landscape of water motion in the access channel, revealed that preferred interaction sites within the channel are separated by small energy barriers (<1.5 kcal/mol). Most importantly, water-bridged conformations lie on a shoulder just 1 kcal/mol above one local minimum and thus are easily accessible. Such an energy landscape appears as a requisite for the effectiveness of compound I formation, whereby the H-bonding pattern involving reactants and catalytic residues (including the intervening water molecule) has to rearrange to deliver the proton to the distal OH moiety of the hydrogen peroxide and thereby lead to heterolytic O-O cleavage. Our study provides an example of a system for which the "reactive configurations" (i.e., structures characterized by a low barrier for the chemical transformation) correspond to a minor population of the system and show how equilibrium molecular dynamics and free-energy calculations may conveniently be used to ascertain that such reactive conformations are indeed accessible to the system. Once again, the MD and QM/MM combination shows that a single water molecule acts as a biocatalyst in the cycle of HRP.

Sulfiredoxin Protects Peroxiredoxin II Overoxidation by Inhibiting Disulfide to Sulfhydryl Regeneration Step in Peroxidase Catalysis

Sulfiredoxin Protects Peroxiredoxin II Overoxidation by Inhibiting Disulfide to Sulfhydryl Regeneration Step in Peroxidase Catalysis

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin: Nα-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.

Thiol Chemistry in Peroxidase Catalysis and Redox Signaling

The oxidation chemistry of thiols and disulfides of biologic relevance is described. The review focuses on the interaction and kinetics of hydrogen peroxide with low-molecular-weight thiols and protein thiols and, in particular, on sulfenic acid groups, which are recognized as key intermediates in several thiol oxidation processes. In particular, sulfenic and selenenic acids are formed during the catalytic cycle of peroxiredoxins and glutathione peroxidases, respectively. In turn, these enzymes are in close redox communication with the thioredoxin and glutathione systems, which are the major controllers of the thiol redox state. Oxidants formed in the cell originate from several different sources, but the major producers are NADPH oxidases and mitochondria. However, a different role of the oxygen species produced by these sources is apparent as oxidants derived from NADPH oxidase are involved mainly in signaling processes, whereas those produced by mitochondria induce cell death in pathways including also the thioredoxin system, presently considered an important target for cancer chemotherapy.

Influence of pH upon Surface‐enhanced Enzyme‐catalyzed Luminol Chemiluminescence at Vicinity of Nanoscale‐corrugated Gold and Silver Films

Au and Ag biochips were fabricated to investigate the influence of pH upon the chemiluminescence (CL) of luminol at vicinity of surface-adsorbed peroxidase. A nanoscaled-corrugation of the metal induces an enhancement of the luminol CL which is maximal in the pH range favoring peroxidase catalysis and greater for gold than for silver. This is the proof that, in the CL process, the reactions involving peroxidase are surface-enhanced near corrugated surfaces.

Biotransformation of Sinapic Acid Catalyzed by Momordica charantia Peroxidase

Biotransformation of sinapic acid (1) with H2O2/Momordica charantia peroxidase, which exists in the widely used food M. charantia, at pH 5.0, 43 degrees C, in the presence of acetone resulted in six compounds, including four new compounds. Compound 2 was the first isolation of a new unique sinapic acid tetrameric derivate, which is formed by peroxidase catalysis in vitro. Besides 2, three other new sinapic acid dimers, 3-5 have also been isolated. Their structures were established on the basis of spectroscopic data. Compound 5 showed a stronger antioxidative activity than the parent sinapic acid (1). Compounds 4 and 5 significantly inhibited the growth of HL-60 cell at the concentration of 10-5 microl/L.

Enzymatic epoxidation and polymerization of cardanol obtained from a renewable resource and curing of epoxide-containing polycardanol

Epoxide-containing polycardanol was enzymatically synthesized via two routes using two different enzymes, viz. lipase and peroxidase. Lipase catalysis was used for the epoxidation of the unsaturated alkyl chains of both cardanol and polycardanol. Peroxidase catalysis was used for the polymerization of both cardanol and epoxide-containing cardanol. One route was the synthesis of epoxide-containing cardanol from cardanol, hydrogen peroxide and an organic acid in the presence of lipase, followed by the polymerization of the phenolic functional groups of cardanol using peroxidase. In the other route, polymerized cardanol was prepared from cardanol and hydrogen peroxide in the presence of peroxidase and, subsequently, the epoxide-containing polycardanol was synthesized from polycardanol, hydrogen peroxide and an organic acid in the presence of lipase. NMR and IR spectroscopy confirmed the polymer structure, and the former route yielded epoxide-containing polycardanol in a higher yield of over 90%. The curing of the resulting polymers proceeded thermally at 150 °C, yielding transparent polymeric films with a high gloss surface within 3 h. The pencil scratch hardness of the present films was improved compared with that of polycardanol. Owing to the epoxide contained in the polymerized cardanol, the film cured with phenalkamine showed a higher hardness value after a relatively short curing time.

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.

Peroxidase‐Catalyzed Coupling of Solid‐Supported ortho‐Methoxyphenols

AbstractEnzyme‐catalyzed CC bond formation has been performed on solid‐supported phenols, thereby expanding the repertoire of enzymatic catalysis on resin‐bound substrates. In the presence of hydroquinone bound to various commercial resins, soybean peroxidase catalyzed the coupling of apocynin (acetovanillone) to give dimers, trimers, and oligomers with structures similar to what is obtained in solution‐phase reactions. In addition to phenolic coupling, peroxidase catalyzed a Fries rearrangement presumably due to rearrangement of the acyl phenolic radicals generated on the solid phase through peroxidase catalysis. This represents the first enzymatic Fries rearrangement reported to date. Together with the solid‐phase biocatalytic phenolic coupling, these results provide a route to the further expansion of enzymatic catalysis in combinatorial library synthesis.