Direct electrochemistry of recombinant tobacco peroxidase on gold

Direct electron transfer (DET) reactions of recombinant tobacco peroxidase (rTOP), namely direct electroreduction of Compound I/Compound II and heme Fe^3+/2+ conversion, were studied on gold electrodes. rTOP of wild type, non-glycosylated, was produced using an Escherichia coli expression system. At pH 5.0, the redox potential for direct electrochemical transformation of the Fe^3+/2+ of the peroxidase heme was −143 mV vs. AgAgCl, and 0.26 ± 0.07 pmol of the adsorbed rTOP were in DET contact with the gold electrode. The total amount of the adsorbed rTOP estimated from QCM data was 53 ± 5 pmol/cm² or 1.67 pmol when referred to the surface area of the electrodes used for electrochemical measurements. Of 1.67 pmol of adsorbed rTOP, only 0.76 pmol were catalytically active. DET between Au and the enzyme was also studied in the reaction of the bioelectrocatalytic reduction of H₂O₂ by cyclic voltammetry and amperometric detection of H₂O₂ at +50 mV with rTOP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Maximal bioelectrocatalytic current response of the rTOP-modified gold electrodes to H₂O₂ was observed at pH 5.0 and stemmed from its bioelectrocatalytic reduction based on DET between Au and the active site of rTOP. Kinetic analysis of the DET reactions gave 52% of the adsorbed rTOP molecules active in DET reactions (0.4 pmol of adsorbed catalytically active rTOP, correspondingly), which correlated well with the non-catalytic-voltammetry data. DET was characterised by a heterogeneous ET rate constant of 13.2 s⁻¹, if one takes into account the QCM data, and 19.6 s⁻¹, if the amount of rTOP estimated from the data on DET transformation of Fe^3+/2+ couple of rTOP is considered. The sensitivity for H₂O₂ obtained for the rTOP-modified Au electrodes was 0.7 ± 0.1 A M⁻¹ cm⁻². These are the first ever-reported data on DET reactions of anionic plant peroxidases on bare gold electrodes.     

Effect of the Components of a Buffer Solution on the Catalytic Activity of the NAD+-Dependent Formate Dehydrogenase from the Bacterium Staphylococcus aureus

Moscow University Chemistry Bulletin - NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from bacterium Staphylococcus aureus (SauFDH) is the most active enzyme among FDHs of this group;...     

Membrane detection of nanogram amounts of formate dehydrogenase

A method for the detection of nanogram amounts of formate dehydrogenase on membranes was developed. The optimization of the method was carried out for determination of the best mediator concentration and tetranitrotetrazolium blue. It was found that 5-methyl-phenazil methosulfate is a more effective mediator than phenazine methosulfate. Different types of membranes were tested and it was shown that nitrocellulose membrane provides a low background and the highest sensitivity. Under optimal conditions the detection limit was 15 ng of the enzyme in the probe.     

The influence of pH on the thermal stability of penicillin acylase from <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

Penicillin G acylase (PA, EC 3.5.1.11) from Alcaligenes faecalis (AfPA) is one of the most thermostable bacterial penicillin acylases. However, systematic data about the thermal stability of AfPA are not found in the literature. A systematic study of the influence of pH on the thermal stability of AfPA was done in the pH range 7.5–9.5. It was found that in all pH ranges studied the enzyme inactivation follows first-order kinetics. The dependence of the inactivation rate constant on pH has an S-shape with an inflection point at pH 8.3–8.5. The temperature dependences of the inactivation rate constant at four pH values were obtained and activation parameters ΔH ^# and ΔS ^# were calculated for each pH value. The decrease of both values, ΔH ^# and ΔS ^# with pH growth shows that a minimum of one iogenic group is essential for the enzyme’s thermal stability.     

Recombinant formate dehydrogenase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: Preparation,crystal growth in microgravity,and preliminary X-ray diffraction study

Crystals of high-purity recombinant NAD⁺-dependent formate dehydrogenase from the higher plant Arabidopsis thaliana (AraFDH) were grown in microgravity in the Modul’-1 protein crystallization apparatus on the International Space Station. The space-grown crystals have larger sizes than those grown on Earth. X-ray diffraction data suitable for determining the three-dimensional structure were collected from the space-grown crystals to a resolution of 1.22 Å using an X-ray synchrotron source. The crystals belong to sp. gr. P4₃2₁2; the unit-cell parameters are a = b = 107.865 Å, c = 71.180 Å, α = β = γ = 90°.     

Three-dimensional structure of laccase from Coriolus zonatus at 2.6 Å resolution

Laccase (oxygen oxidoreductase, EC 1.14.18.1) belongs to the copper-containing oxidases. This enzyme catalyzes reduction of molecular oxygen by different organic and inorganic compounds to water without the formation of hydrogen peroxide. The three-dimensional structure of native laccase from Coriolus zonatus was solved and refined at 2.6 Å resolution (R _factor = 21.23%, R _free = 23.82%, rms deviations for the bond lengths and bond angles are 0.008 Å and 1.19°, respectively). The primary structure of the polypeptide chain and the architecture of the active site were refined. The carbohydrate component of the enzyme was identified. The access and exit water channels providing the access of molecular oxygen to the active site and release of water, which is the reduction product of molecular oxygen, from the protein molecule were found in the structure.     

Structural optimization of adaptaquin,a HIF prolyl hydroxylase inhibitor

Russian Chemical Bulletin - The structural optimization of adaptaquin, viz., 7-(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methylquinolin-8-ol, a HIF (hypoxia inducible factor) prolyl hydroxylase...