Anionic Peroxidase Research Articles

Influences of Ipomoea batatas Anti-Cancer Peptide on Tomato Defense Genes.

This study investigates the impact of IbACP (Ipomoea batatas anti-cancer peptide) on defense-related gene expression in tomato leaves, focusing on its role in plant defense mechanisms. Previously, IbACP was isolated from sweet potato leaves, and it was identified as a peptide capable of inducing an alkalinization response in tomato suspension culture media. Additionally, IbACP was found to regulate the proliferation of human pancreatic adenocarcinoma cells. Elucidate IbACP's molecular influence on defense-related gene expression in tomato leaves using next-generation sequencing analysis. To assess the impact of IbACP on defense-related gene expression, transcriptome data were analyzed, encompassing various functional categories such as photosynthesis, metabolic processes, and plant defense. Semi-quantitative reverse-transcription polymerase chain reaction analysis was employed to verify transcription levels of defense-related genes in tomato leaves treated with IbACP for durations ranging from 0 h (control) to 24 h. IbACP induced jasmonic acid-related genes (LoxD and AOS) at 2 h, with a significant up-regulation of salicylic acid-dependent gene NPR1 at 24 h. This suggested a temporal antagonistic effect between jasmonic acid and salicylic acid during the early hours of IbACP treatment. Downstream ethylene-responsive regulator genes (ACO1, ETR4, and ERF1) were consistently down-regulated by IbACP at all times. Additionally, IbACP significantly up-regulated the gene expressions of suberization-associated anionic peroxidases (TMP1 and TAP2) at all time points, indicating enhanced suberization of the plant cell wall to prevent pathogen invasion. IbACP enhances the synthesis of defense hormones and up-regulates downstream defense genes, improving the plant's resistance to biotic stresses.

A new versatile peroxidase with extremophilic traits over-produced in MicroTom cell cultures

Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H2O2. In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70–80 °C and the presence of NaCl (1.0–4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0–7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0–7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry.

Heterologous production and secretion of active-form plant peroxidase in Brevibacillus choshinensis and their monolignol polymerization abilities analysis

Plant peroxidases are important for several processes, such as defense against pathogens, and auxin metabolism. In this study, we report the active production and secretion of plant peroxidase and mutated enzymes in the bacterium Brevibacillus choshinensis for the first time in the world. We introduced mutations into prxA3a, an anionic peroxidase gene of hybrid aspen, Populus × kitakamiensis, to substitute the amino acid residues at the surface of the protein and analyzed their modified substrate specificities. We have also reported heterologous expression of PrxA3a and mutated enzymes in yeast. Enzymes secreted in the culture medium by B. chosinensis were purified by Ni affinity chromatography, anion-exchange chromatography, and size-exclusion chromatography. The ability of the mutated enzymes to polymerize sinapyl alcohol, a monolignol, was higher than that of the wild-type enzyme. In particular, the FYAW-mutated enzyme produced by the bacterium showed higher polymerization activity, similar to that of the FYAW-mutated enzyme produced by yeast.

Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots.

Peroxidase is a commonly used enzyme with a wide range of applications. Horseradish (Armoracia rusticana) is the most well-known source of peroxidase enzyme. Peroxidases extracted from other plant sources have also been proved as useful, sometimes even superior, comparing to traditional horseradish peroxidase (HRP). In the present study, two novel peroxidase isoenzymes were purified and characterized from Raphanus sativus L. var niger roots. Two anionic peroxidase isoenzymes were purified using diafiltration, ammonium sulfate precipitation, DEAE anion-exchange chromatography, and concanavalin A affinity chromatography. The heaviest anionic isoenzyme (isoenzyme A) has a MW of about 110 KD, and the other anionic isoenzyme (isoenzyme B) has a MW of 97 KD. Both isoenzymes have an optimum temperature of 40°C, but the activity of isoenzyme B is much more dependent on temperature with a Q10 of 3.5, while isoenzyme A has a Q10 of 1.7. These isoenzymes showed great thermal stability at 37°C and 4°C. Isoenzyme A showed the highest activity at pH 5 and it was found to be more stable at pH 6, whereas isoenzyme B showed the highest activity at pH 6 and is more stable at pH 7. Isoenzyme A has a Km value of 10.63mM and 0.043mM, and isoenzyme B has a Km of 15.38mM and 0.067mM for 4-aminoantipyrine and H2O2, respectively. The isoenzymes purified from Raphanus sativus L. var niger offer excellent chemical and thermal stability, which encourages further studies on their suitability for biotechnological applications.

Purification and characterization of peroxidases from garden cress sprouts and their roles in lignification and removal of phenol and p-chlorophenol.

The study aims to evaluate the relation between peroxidases of day-6 garden cress sprouts and phenolic compounds. Three cationic, three anionic, and two unbounded peroxidases were separated from day-6 garden cress sprouts. Cationic (GCP1) and anionic (GCP2) peroxidases were purified with molecular masses of 25 and 40kDa, respectively. The Km values of GCP1 toward H2 O2 and guaiacol were lower than GCP2. The anionic GCP2 exhibited high affinity toward some lignin monomers, sinapyl alcohol, coniferyl alcohol, cinnamic and ferulic acids. Therefore, GCP2 is considered as a lignin peroxidase and contributed in lignin synthesis. The activity of GCP1 and GCP2 was stable at a wide pH range 5.5-8.0 and 6.0-7.5, respectively. Both peroxidases showed the same thermal stability range 20-50°C. GCP2 was more resistant against the effect of metal ions than GCP1. GCP2 showed high ability to remove of phenol and p-chlorophenol from effluent compared to GCP1. PRACTICAL APPLICATIONS: Generally, garden cress is used as a test plant to conduct biomonitoring of pollution in urban soil on a wide scale because of its simplicity, sensitivity, and cost-effectiveness. Peroxidase is an important antioxidant enzyme, which elevated when plant subjected to pollution. Recently, we reported that the increase of peroxidase activity was strongly correlated with high phenolic content and antioxidant activity during the germination of garden cress. In the present study, anionic peroxidase GCP2 may play an important role in lignification process and removal of phenol and p-chlorophenol from polluted soil/wastewater as well as resisted the harmful effect of heavy metals. Cationic peroxidase GCP1, as a natural scavenger, had high affinity toward H2 O2 coupled to oxidation of some plant phenolic compounds suggesting its role in consuming of excess H2 O2 .

StPOPA, encoding an anionic peroxidase, enhances potato resistance against Phytophthora infestans

Late blight, caused by Phytophthora infestans, is the most devastating potato disease worldwide and results in catastrophic production losses. Reactive oxygen species (ROS) are signal molecules utilized at the early stage of plant immunity. Specifically, anionic peroxidases are reported to be involved in many plants’ defense systems. However, there is limited information about the function of anionic peroxidase genes in potato immunity. Here, we report that StPOPA, a gene encoding a suberization-associated anionic peroxidase, was induced by P. infestans infection, mechanical damage, as well as jasmonic acid and ethylene treatments. Overexpression of StPOPA gene in potato enhanced potato resistance against P. infestans by promoting the accumulation of callose in the cell wall and ROS in the cytoplasm, and then the callose and ROS restricted the infection and spreading of the disease possibly by purposeful programmed cell death. Our results suggest that StPOPA plays a positive role in potato resistance to immunochallenge, and this gene could be used for the genetic improvement of resistance against potato late blight.

Эндофитные бактерии Bacillus subtilis 26Д снижают пораженность растений картофеля фитофторозом, стимулируя транскрипционную активность жасмонат-зависимых генов

Bacillus subtilis 26D stimulated transcriptional activity of jasmonic acid biosynthesis genes and jasmonate-dependent protective genes, including anionic peroxidase involved in lignification, which increased their resistance to late blight pathogen.

Cloning and functional analysis of the promoter of a stress-inducible gene (Zmap) in maize.

The anionic peroxidases play an important role in a variety of plant physiological processes. We characterized and isolated the Zmap promoter (PZmap) at the 5′ flanking region in order to better understand the regulatory mechanisms of Zmap gene expression. A series of PZmap deletion derivatives, termed a1 –a6, at positions −1694, −1394, −1138, −784, −527 and −221 from the translation start site were blended to the β-glucuronidase reporter gene. Agrobacterium-mediated transformation method was used to study each deletion construct in tobaccos. Sequence analysis showed that several cis-acting elements (MYB binding site, Box-II, a TGACG-element, a CGTCA-element and a low temperature responsive element) were located within the promoter. Deletion analysis suggested the sequence between −1,694 and −1394bp may contain cis-elements associated with GUS up regulation. The MYB binding site (-757) might act as a negative drought-responsive element. There might be repressor elements located in the region (−1,694 to −1394bp) to repress Zmap expression under 4°C. The characterized promoter would be an ideal candidate for genetic engineering for improving the resistance of maize to different stressors.

Simultaneous detection of three biomarkers related to acute myocardial infarction based on immunosensing biochip

An immunosensing biochip for simultaneous detection of three biomarkers related to acute myocardial infarction (AMI) was developed based on anionic soybean peroxidase (SBP) functionalized nanoprobe and chemiluminescent imaging. The nanoprobes (Ab2-SiO2-SBP) were fabricated by co-immobilization of SBP and one of the detection polyclonal antibodies, anti-cardiac troponin I antigen (anti-cTnI), anti-creatine kinase-MB (anti-CK-MB) and anti-myoglobin (anti-Myo), on the silica nanoparticle surface. The detection sensitivity was enhanced since the large surface area of silica carriers increased the loading of SBP for per sandwiched immunoreaction. The immunosensing biochip designed as 3 × 8 wells array was constructed by simultaneously immobilizing three capture monoclonal antibodies on the same one microtiter well with 2 × 3 active spots. In the presence of target protein, the nanoprobes will be attached onto the spots with high specificity through the sandwiched immunoreactions, which triggered the chemiluminescence (CL) signals on each sensing site of the microtiter plates and allowed to CL imaging of three biomarkers in one well at the same time. Therefore, the proposed biochip was a promising convenient strategy for simultaneous detection of cTnI, CK-MB and Myo, which showed potential application for multianalyte determination in clinical diagnostics.

The reinforcement of potato cell wall as part of the phosphite-induced tolerance to UV-B radiation

Phosphites (Phis), inorganic salts of phosphorous acid, have shown to be effective in protection of plants against biotic stress. Recently, we have described that potassium phosphite (KPhi) induces tolerance to UV-B radiation in potato. To counteract the harmful effect of UV radiation, plants accumulate UV-screening compounds, such as flavonoids, sinapate ester, and lignin. In previous work, we have shown an increase in guaiacol peroxidase (POD) activity in plants pretreated with KPhi and further exposed to UV-B radiation. In order to continue with this study, the expression of different enzymes and components involved in cell wall reinforcement were analyzed. An isoform of POD induced by KPhi was analyzed by isoelectric focusing and further identified as suberization-associated anionic peroxidase (POPA) by mass spectrometry. In addition, other enzymes participating in lignin biosynthesis, like caffeoyl-CoA O-methyltransferase (CCoAOMT), determined by accumulation of transcripts, and laccase activity, visualized in zymogrames, were increased by KPhi treatment previous to UV-B exposure. Further, the accumulations of extensin (EXT) transcripts and of conjugated polyamines (PAs) were increased by KPhi treatment previous to UV-B exposure. All these results suggest cell wall reinforcement in leaves due to KPhi pretreatment followed by UV-B exposure.

Highly sensitive, stable and selective hydrogen peroxide amperometric biosensors based on peroxidases from different sources wired by Os-polymer: A comparative study

A comparison was made between two plant peroxidases, cationic horseradish peroxidase (HRP) and anionic tobacco peroxidase (TOP), combined with a highly cationic osmium polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2poly(N-vinylimidazole)10Cl]+2/+ ([Os(dmp)PVI]+/2+) to develop highly sensitive, stable and selective hydrogen peroxide biosensors. The two different plant peroxidases were individually immobilized onto graphite rod (G) electrodes by a three steps drop-casting procedure consisting of the subsequent deposition of an aqueous solution of ([Os(dmp)PVI]+/2+), followed by a solution of poly(ethyleneglycol) diglycidyl ether (PEGDGE), used as a cross linking agent and finally an aliquot of a solution of cationic HRP or anionic TOP to make HRP/PEGDGE/[Os(dmp)PVI]+/2+/G and TOP/PEGDGE/[Os(dmp)PVI]+/2+/G based electrodes, respectively. Electrochemical experiments were carried out to investigate the influence of the surface charge of the enzyme and the charge of the polymer on the efficiency of the electron transfer (ET) between the enzyme and the wiring redox polymer and the efficiency for electrocatalytic reduction of H2O2. In the case of HRP a decrease in the ET rate was observed due to the repulsion between this enzyme and the polymer, both positively charged, whereas with TOP there was an enhanced ET rate due to the attraction between the anionic enzyme and the cationic polymer. The effects of enzyme loading and pH were investigated. Both peroxidase modified electrodes exhibited a wide dynamic response range (1–500μM H2O2) and a low detection limit (0.3μM H2O2). The TOP based electrode showed a higher sensitivity (470nAμM−1cm−2) compared to that of the HRP based electrode (300nAμM−1cm−2) and an improved long-term stability (decrease in 17.3% upon 30days compared with 50% for HRP). Both enzyme electrodes showed a response time of 3s. The HRP based sensor was more sensitive to the presence of phenolic compounds acting as alternative electron donors, whereas the TOP based sensor was virtually interference free. Both HRP and TOP based electrodes were successfully tested in contact lens cleaning samples and real “spiked” samples from different sources such as tap water, milk and dairy products.

Peroxidases from alfalfa roots: Catalytic properties and participation in degradation of polycyclic aromatic hydrocarbons

From the roots and root exudates of 3-week-old plants of alfalfa (Medicago sativa L.), anionic and cationic peroxidases differing in principal physicochemical and catalytic properties were isolated and purified. Main features of anionic peroxidases detected in the roots and root exudates were identical. Phenanthrene present in the soil used for alfalfa growing influenced the number of forms and activity of peroxidases in crude enzyme preparations but did not affect the properties of pure enzymes. In the presence of a synthetic mediator, purified peroxidases can oxidize phenanthrene and its derivatives, including potential microbial metabolites of polycyclic aromatic hydrocarbons (PAH). The fact that the enzymes excreted in root exudates in a purified form can oxidase PAH proves their participation in degradation of PAH and their microbial metabolites in alfalfa rhizosphere. These new data indicate that the processes of plant and microbial degradation of pollutants in the rhizosphere are coupled; they are relevant to understanding the molecular mechanisms of degradation of persistent pollutants by plant-microbial complexes.

Verticillium Ave1 effector induces tomato defense gene expression independent of Ve1 protein

ABSTRACTVerticillium resistance is thought to be mediated by Ve1 protein, which presumably follows a “gene-for-gene” relationship with the V. dahliae Ave1 effector. Because in planta analyses of Ave1 have relied so far on transient expression of the gene in tobacco, this study investigated gene function using stably expressing 35S:Ave1 transgenic tomato. Transgenic Ave1 expression was shown to induce various defense genes including those coding for PR-1 (P6), PR-2 (βbeta-1,3-glucanase) and peroxidases (anionic peroxidase 2, Cevi16 peroxidase). Since a Ve1− tomato cultivar served as germplasm, these results indicate that Ave1 induces these defense genes independently of Ve1.

Induced expression of Serratia marcescens ribonuclease III gene in transgenic Nicotiana tabacum L. cv. SR1 tobacco plants

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.

Inhibition of IAA oxidase activity of wheat anionic peroxidase by chitooligosaccharides

The study demonstrated that chitooligosaccharides with a molecular weight of 5–10 kDa and a degree of acetylation of 65% exhibited an auxin-like effect in wheat plants and also played an important role in regulating the activity of polysaccharide (chitin)–specific anion isoenzymes of peroxidase oxidizing indole acetic acid. Changes in the kinetic parameters of the interaction of the wheat anionic chitin-specific peroxidase with pI ∼3.5 with chitin oligomers in the presence of indoleacetic acid were pH-depended and indicated that chitooligosaccharides significantly impair the ability of the enzyme for oxidation at pH levels of 4.2 and 6.0. It can be assumed that chitooligosaccharides not only induce protective plant systems but also increase the accumulation of auxin in plant tissues, thus adversely affecting a number of components of the plant protective system against pathogens.

Enhanced resistance to late blight pathogen conferred by expression of rice oxalate oxidase 4 gene in transgenic potato

Phytophthora infestans is the pathogen responsible for late blight disease of potato. Multiple spraying of crop protection chemicals is so far the only means to control the disease. Despite the continuous effort from breeders, late blight resistant potato varieties are not available to farmers. In this context, development of genetic resistance by incorporating new genes through genetic engineering is an attractive way to combat the pathogen. Here, we report the development of transgenic potato cultivars overexpressing the rice oxalate oxidase 4 gene (Osoxo4). Transgenic lines showed higher activity of oxalate oxidase enzyme and were able to degrade externally applied oxalic acid. The levels of reactive oxygen H2O2 were elevated in transgenic plants. The transcription of defense related genes (anionic peroxidase and phenylalanine ammonia lyase) was found to be elevated after pathogen inoculation. Detached leaf bioassays with spore suspension and in vitro plantlet bioassays with mycelia of P. infestans showed enhanced resistance in transgenic plants. Interestingly, constitutive overexpression of Osoxo4 did not show any effect on the morphology and yield of transgenic potato plants as evident from agronomic performance studies. Thus, constitutive expression of Osoxo4 represents an efficient strategy for engineering late blight resistance in potato.

A cell wall-bound anionic peroxidase, PtrPO21, is involved in lignin polymerization in Populus trichocarpa

Class III peroxidases are members of a large plant-specific sequence-heterogeneous protein family. Several sequence-conserved homologs have been associated with lignin polymerization in Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum, Zinnia elegans, Picea abies, and Pinus sylvestris. In Populus trichocarpa, a model species for studies of wood formation, the peroxidases involved in lignin biosynthesis have not yet been identified. To do this, we retrieved sequences of all PtrPOs from Peroxibase and conducted RNA-seq to identify candidates. Transcripts from 42 PtrPOs were detected in stem differentiating xylem (SDX) and four of them are the most xylem-abundant (PtrPO12, PtrPO21, PtrPO42, and PtrPO64). PtrPO21 shows xylem-specific expression similar to that of genes encoding the monolignol biosynthetic enzymes. Using protein cleavage-isotope dilution mass spectrometry, PtrPO21 is detected only in the cell wall fraction and not in the soluble fraction. Downregulated transgenics of PtrPO21 have a lignin reduction of ~20 % with subunit composition (S/G ratio) similar to wild type. The transgenics show a growth reduction and reddish color of stem wood. The modulus of elasticity (MOE) of the stems of the downregulated PtrPO21-line 8 can be reduced to ~60 % of wild type. Differentially expressed gene (DEG) analysis of PtrPO21 downregulated transgenics identified a significant overexpression of PtPrx35, suggesting a compensatory effect within the peroxidase family. No significant changes in the expression of the 49 P. trichocarpa laccases (PtrLACs) were observed.

Biochemical, anatomical and molecular level changes in cucumber (Cucumis sativus) seedlings exposed to copper oxide nanoparticles

AbstractThe effect of copper oxide nanoparticles (CuONPs) at the biochemical, anatomical and molecular level was investigated in cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in semi-solid half strength Murashige and Skoog medium supplemented with 0, 50, 100, 200, 400 and 500 mg/L of CuONPs for fifteen days under controlled growth chamber conditions. The results showed that exposure to all concentrations of CuONPs resulted in significant reduction in shoot and root growth and biomass. A concentration-dependant increase in reactive oxygen species (ROS) generation, malondialdehyde production, lignin content and decline in mitochondrial membrane potential were observed. Cross-sections of stem showed anatomical changes, viz. an increase in xylogenesis in CuONPs exposed plants. Significant modulation in the expression of genes related to oxidative stress and lignin biosynthesis, i.e. catalase, ascorbate peroxidase, phenylalanine ammonia lyase, cinnamate 4-hydroxylase, anionic and cationic peroxidases were observed in shoots and roots as a result of CuONPs exposure. Taken together, exposure to CuONPs has resulted in excess ROS generation, lignification and growth suppression in Cucumis sativus seedlings.

High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli

Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8μg/mL solubilized rTOP protein and the refolding medium containing 0.3mM oxidized glutathione, 0.05mM dithiothreitol, 5mM CaCl2, 5% glycerol in 50mM Tris–HCl buffer, pH 9.6, with 1μM hemin added at the 24th hour of incubation. A practically important discovery was a 30–40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6mg per L of E. coli culture).

Site-directed mutagenesis of tobacco anionic peroxidase: Effect of additional aromatic amino acids on stability and activity.

Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis.