Search

• Electrochemical property of FDH lacking a cytochrome subunit (ΔC FDH) was studied. • DET of ΔC FDH was observed at porous Au electrodes modified with thiols. • Electrode-active site of ΔC FDH was estimated by kinetic analysis of DET signals. • Uncharged aromatic thiols on porous Au electrodes enhance DET of ΔC FDH. • An enhancement mechanism for DET by aromatic thiols was proposed. A heterotrimeric membrane-bound d -fructose dehydrogenase (FDH) from Gluconobacter japonicus , composed of subunits L, C, and S, shows strong direct electron transfer (DET)-type bioelectrocatalytic performance in the two-electron oxidation of d -fructose. The electrode-active site of the enzyme is the heme c moiety in the subunit C. A previous study reported the construction of the subunit L/S subcomplex (ΔC FDH) lacking the subunit C. In this work, we attempted to realize the DET-type reaction of ΔC FDH using porous gold electrodes functionalized with several thiols, and investigated the influence of the thiols on DET. Kinetic analysis of the steady-state catalytic waves revealed that the electrode-active site of ΔC FDH is the [3Fe-4S] iron-sulfur cluster in the L subunit. The experimental results demonstrated that uncharged aromatic thiols on the electrode enhance the DET-type reaction of ΔC FDH. Based on the pH-dependent profile of the DET-type activity and the surface conditions of the modified thiols evaluated by electrochemical reductive desorption, we suggest an enhancement mechanism for DET by aromatic thiols. [ABSTRACT FROM AUTHOR]

The effect of mutation on the redox potentials (E°′) of the heme moieties in the variants of d-fructose dehydrogenase (FDH) was investigated by mediated spectroelectrochemical titrations. The replacement of the axial ligand of heme from methionine to glutamine changes the E°′ value more negatively than that of the corresponding heme moiety in the recombinant (native) FDH (rFDH). The determined E°′ values of non-targeted heme moieties in the variants were also shifted in a negative direction from that in rFDH. Thus, enzyme modification changes E°′ of the heme moieties in unmodified protein regions.

• Critical amino acid residues of FDH in catalysis were predicted. • Four FDH variants were evaluated using electrochemistry and structural biology. • Biocatalytic mechanisms of FDH have been elucidated. • Structures of in-silico variants were validated by comparing them to cryo-EM structures. • Relative activity of variants with D-tagatose was improved over that of rFDH. D-Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of intense direct electron transfer (DET)-type bioelectrocatalysis, was investigated using protein engineering, electrochemistry, structural biology, and bioinformatics. DET-type reactions have led to an increase in energy efficiency, biocompatibility, and design freedom in bioelectrochemical devices, such as biofuel cells, biosensors, biosupercapacitors, and bioreactors. However, one of the greatest challenges for this reaction is the limited number of the applicable enzymes. The creation of variants with altered substrate specificities using template enzymes is a promising solution to address this problem. For creating variants using FDH as a template, it is important to understand the biocatalytic mechanisms. Recently, the structure of FDH has been analyzed using cryo-electron microscopy (cryo-EM) single particle analysis, and the three amino acid residues (N1146, H1147, and N1190) may be critical for substrate recognition by FDH. In this study, we aimed to understand the mechanisms of the biocatalytic reaction of FDH and discuss the roles of these residues. In addition, we validated the structures of the in-silico variants by comparing them to the cryo-EM structures. Furthermore, for the variants of N1146 involved in D-fructose recognition, we have verified the changes in specificity for other substrates. [ABSTRACT FROM AUTHOR]

Abstract: The catalytic activity of a membrane-bound enzyme, d-fructose dehydrogenase (FDH), at the polarized oil/water (O/W) interface was studied. Multisweep cyclic voltammetry and ac voltammetry were carried out to show the irreversible adsorption of FDH at the interface. Using the thusly prepared FDH-adsorbed O/W interface, clear steady-state catalytic current was observed in amperometry and cyclic voltammetry, where 1,1′-dimethylferrocenium ion (DiMFc+, electron acceptor) and d-fructose (substrate) were added to the O and W phases, respectively. The observed catalytic current was then analyzed by using two mechanisms. In mechanism (A), the heme c site of FDH, where DiMFc+ is reduced, was assumed to be located in the O-phase side of the interface. The intramolecular electron transfer in FDH should be affected by the Galvani potential difference of the interface (). However, the theoretical equations derived for the catalytic current could not reproduce the experimental data. In mechanism (B), the heme c site was assumed to be in the W-phase side. In this case, should affect the interfacial distribution of DiMFc+. This mechanism could reproduce well the observed potential dependence of the catalytic current. [Copyright &y& Elsevier]

Abstract: The aim of this study was to develop a simple, rapid and highly sensitive sensor for measuring the rare sugar d-psicose. The proposed system adopts amperometric flow analysis and two consecutive enzyme reactions consisting of a reactor packed with d-tagatose 3-epimerase (DTE)-immobilized beads, which converts d-psicose to d-fructose, and a carbon-paste electrode containing d-fructose dehydrogenase (DFDH). In order to fabricate a robust sensor system, various experimental parameters were optimized including the buffer composition, flow rate for the two enzyme reactions and the size of micro-flow cell. The developed sensor responded linearly to d-psicose concentration in the range from 0.08 to 50mM (R 2 =0.988). The signal/noise ratio was 3.0 for the 0.08mM d-psicose solution, and the relative standard deviations were 1.7 (n =20) and 2.6% (n =20) for the 10 and 20mM d-psicose solutions, respectively. One round of assay was completed within 8min. Our results suggest that the sensor can be used not only for the detection of d-psicose in food samples but also for monitoring d-psicose within the environment. Moreover, the sensor system can be applied to the detection of many other rare sugars by using the same measurement principle. [Copyright &y& Elsevier]

Abstract: Direct electrochemistry of heme multicofactor-containing enzymes, e.g., microbial theophylline oxidase (ThOx) and d-fructose dehydrogenase (FDH) from Gluconobacter industrius was studied on alkanethiol-modified gold electrodes and was compared with that of some previously studied complex heme enzymes, specifically, cellobiose dehydrogenase (CDH) and sulphite oxidase (SOx). The formal redox potentials for enzymes in direct electronic communication varied for ThOx from −112 to −101 mV (vs. Ag|AgCl), at pH 7.0, and for FDH from −158 to −89 mV, at pH 5.0 and pH 4.0, respectively, on differently charged alkanethiol layers. Direct and mediated by cytochrome c electrochemistry of FDH correlated with the existence of two active centres in the protein structure, i.e., the heme and the pyrroloquinoline quinone (PQQ) prosthetic groups. The effect of the alkanethiols of different polarity and charge on the surface properties of the gold electrodes necessary for adsorption and orientation of ThOx, FDH, CDH and SOx, favourable for the efficient electrode–enzyme electron transfer reaction, is discussed. [Copyright &y& Elsevier]

A new mediated carbon nanotube paste (CNTP) amperometric biosensor for fructose is described. The biosensor is formed by a CNTP electrode modified with an electropolymerized film of 3,4-dihydroxybenzaldehyde (3,4-DHB) and is based on the activity of a commercial available D-fructose dehydrogenase (FDH) immobilized on an immobilon membrane placed on the top of the electrode surface. Analytical parameters such as enzyme immobilization, pH, temperature, and probe lifetime were studied and optimized. The biosensor response current was directly proportional to D-fructose concentration from 5 × 10−6 to 2 × 10−3 mol/L with a detection limit of 1 × 10−6 mol/L and a good reproducibility (RSD = 1.8%, n = 5). The biosensor was used for the determination of fructose content in three honey samples and validated with a commercial spectrophotometric enzymatic kit. [ABSTRACT FROM AUTHOR]

An electrochemical d -fructose sensor was fabricated by immobilizing a variant of d -fructose dehydrogenase on a porous gold microelectrode. The enzyme-modified electrode bioelectrocatalytically oxidizes d -fructose via direct electron transfer. The catalytic current reaches a steady-state limiting value at 0.05 V vs. Ag|AgCl (sat. KCl). The temperature dependence of the sensor suggests that its response is limited by the diffusion of d -fructose. Therefore, the sensor requires no calibration. The sensitivity of the sensor, (2.0 ± 0.2) × 102 μA cm−2 mM−1, is reproducible. The upper limit of detection is ~2.0 mM and the kinetics of the bioelectrocatalytic reaction is determined the limitation. The fabricated sensors were applied in the determination of d -fructose in commercial beverages as well as honey, and compared favorably with a more commonly used photometric method. [ABSTRACT FROM AUTHOR]