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71.
Hushpulian DM Poloznikov AA Savitski PA Rozhkova AM Chubar TA Fechina VA Orlova MA Tishkov VI Gazaryan IG Lagrimini LM 《Biological chemistry》2007,388(4):373-380
The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals. 相似文献
72.
73.
Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed. 相似文献
74.
Stirnimann CU Rozhkova A Grauschopf U Böckmann RA Glockshuber R Capitani G Grütter MG 《Journal of molecular biology》2006,358(3):829-845
Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD. 相似文献
75.
Y. P. Vinetsky A. M. Rozhkova A. M. Chulkin A. D. Satrutdinov O. A. Sinitsyna E. A. Fedorova A. O. Bekkarevich O. N. Okunev A. P. Sinitsyn 《Biochemistry. Biokhimii?a》2009,74(8):882-887
The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator. 相似文献
76.
Sinitsyna OA Fedorova EA Vakar IM Kondratieva EG Rozhkova AM Sokolova LM Bubnova TM Okunev ON Chulkin AM Vinetsky YP Sinitsyn AP 《Biochemistry. Biokhimii?a》2008,73(1):97-106
Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder. 相似文献
77.
The new possibilities of using scattered x-ray radiation in mammology are presented by the example of obtaining the new type of a highly informative breast image, by identifying the distribution of an effective atomic number by dual-energy exposure. 相似文献
78.
79.
Semin IuA Zhavoronkov LP Voron'ko IaV Shvartsburg LK Rozhkova OM 《Radiatsionnaia biologiia, radioecologiia / Rossi?skaia akademiia nauk》2003,43(5):524-527
The work presents results of the experimental study on thymus changes developing after whole-body exposure of mice to ultralow power pulse-modulated electromagnetic field (carrying frequency 2.39 GHz, modulating pulses with frequency 4 Hz, duration of impulses 0.025 sec, average power density 60 mW/cm2, absorbed dose 0.086 J/g or 0.172 J/g). It was shown that a percent of the microwave induced increase or decrease of thymus mass and the number of cells in the organ (y) are determined by the initial mass or number of cells in thymus accordingly to equation of linear regression: (yx = 215-2.25x, where x is the thymus mass of control animals (in a range 31-63 mg) and (yx = 178.6-41x, where x is the initial number of cells in thymus (in a range 0.6 x 10(8)-2.6 x 10(8)) reduced by a factor of 10(8). 相似文献
80.