Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

We have employed computational approaches—FireProt and FRESCO—to predict thermostable variants of the reductase component (C1) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6–5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH−) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300–500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications

Magnetic field effects as a result of the radical pair mechanism are unlikely in redox enzymes

Environmental exposure to electromagnetic fields is potentially carcinogenic. The radical pair mechanism is considered the most feasible mechanism of interaction between weak magnetic fields encountered in our environment and biochemical systems. Radicals are abundant in biology, both as free radicals and reaction intermediates in enzyme mechanisms. The catalytic cycles of some flavin-dependent enzymes are either known or potentially involve radical pairs. Here, we have investigated the magnetic field sensitivity of a number of flavoenzymes with important cellular roles. We also investigated the magnetic field sensitivity of a model system involving stepwise reduction of a flavin analogue by a nicotinamide analogue—a reaction known to proceed via a radical pair. Under the experimental conditions used, magnetic field sensitivity was not observed in the reaction kinetics from stopped-flow measurements in any of the systems studied. Although widely implicated in radical pair chemistry, we conclude that thermally driven, flavoenzyme-catalysed reactions are unlikely to be influenced by exposure to external magnetic fields

Glucose oxidation using oxygen resistant pyranose-2-oxidase for biofuel cell applications

In this study, the effect of oxygen on glucose oxidation using Glucose Oxidase (GOx) and oxygen resistant Pyranose-2-Oxidase (P2O) has been studied. Enzyme solutions with ferrocene carboxylic acid (FcCOOH) as electron mediator were tested with glassy carbon electrode (GCE) under air and nitrogen saturated conditions in a three electrode electrochemical cell system. Electrochemical characterization of enzymes has been achieved in solution by using cyclic voltammetry (CV), linear sweep voltammetry (LSV) and chronoamperometry (CA). In the presence of glucose, CV and LSV results show increasing anodic peak current and decreasing cathodic peak current with increasing glucose concentrations, which reflects the ferrocene-mediated bioelectrocatalysis of glucose oxidation. The experiments with CA show enhanced stability with oxygen resistant P2O where GOx loses 30 % of its current density in the presence of oxygen after 3 hours. These results indicate that P2O, a promising enzyme with no oxygen reactivity and long stability, which can be used in enzymatic biofuel cell applications as an alternative to GOx

Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M–1 s–1. In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s–1. At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is 51 s–1, whereas without 3-HB, the rate constant is 0.43 s–1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme–product (2,5-DHB) complex, with a rate constant of 45 ± 2 s–1. The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em0 for the enzyme–substrate complex is -179 ± 1 mV compared to an Em0 of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme–ligand complex to a fully activated form before flavin reduction takes plac

Enzymatic fuel cells with an oxygen resistant variant of pyranose-2-oxidase as anode biocatalyst

In enzymatic fuel cells (EnFCs), hydrogen peroxide formation is one of the main problems when enzymes, such as, glucose oxidase (GOx) is used due to the conversion of oxygen to hydrogen peroxide in the catalytic reaction. To address this problem, we here report the first demonstration of an EnFC using a variant of pyranose-2-oxidase (P2O-T169G) which has been shown to have low activity towards oxygen. A simple and biocompatible immobilisation approach incorporating multi-walled-carbon nanotubes within ferrocene (Fc)-Nafion film was implemented to construct EnFCs. Successful immobilisation of the enzymes was demonstrated showing 3.2 and 1.7-fold higher current than when P2O-T169G and GOx were used in solution, respectively. P2O-T169G showed 25% higher power output (maximum power density value of 8.45 ± 1.6 μW cm−2) and better stability than GOx in aerated glucose solutions. P2O-T169G maintained > 70% of its initial current whereas GOx lost activity > 90% during the first hour of 12 h operation at 0.15 V (vs Ag/Ag+). A different fuel cell configuration using gas-diffusion cathode and carbon paper electrodes were used to improve the power output of the fuel cell to 29.8 ± 6.1 µW cm−2. This study suggests that P2O-T169G with low oxygen activity could be a promising anode biocatalyst for EnFC applications

Proton-Coupled Electron Transfer and Adduct Configuration Are Important for C4a-Hydroperoxyflavin Formation and Stabilization in a Flavoenzyme

Determination of the mechanism of dioxygen activation by flavoenzymes remains one of the most challenging problems in flavoenzymology for which the underlying theoretical basis is not well understood. Here, the reaction of reduced flavin and dioxygen catalyzed by pyranose 2-oxidase (P2O), a flavoenzyme oxidase that is unique in its formation of C4a-hydroperoxyflavin, was investigated by density functional calculations, transient kinetics, and site-directed mutagenesis. Based on work from the 1970s–1980s, the current understanding of the dioxygen activation process in flavoenzymes is believed to involve electron transfer from flavin to dioxygen and subsequent proton transfer to form C4a-hydroperoxyflavin. Our findings suggest that the first step of the P2O reaction is a single electron transfer coupled with a proton transfer from the conserved residue, His548. In fact, proton transfer enhances the electron acceptor ability of dioxygen. The resulting ·OOH of the open-shell diradical pair is placed in an optimal position for the formation of C4a-hydroperoxyflavin. Furthermore, the C4a-hydroperoxyflavin is stabilized by the side chains of Thr169, His548, and Asn593 in a “face-on” configuration where it can undergo a unimolecular reaction to generate H₂O₂ and oxidized flavin. The computational results are consistent with kinetic studies of variant forms of P2O altered at residues Thr169, His548, and Asn593, and kinetic isotope effects and pH-dependence studies of the wild-type enzyme. In addition, the calculated energy barrier is in agreement with the experimental enthalpy barrier obtained from Eyring plots. This work revealed new insights into the reaction of reduced flavin with dioxygen, demonstrating that the positively charged residue (His548) plays a significant role in catalysis by providing a proton for a proton-coupled electron transfer in dioxygen activation. The interaction around the N5-position of the C4a-hydroperoxyflavin is important for dictating the stability of the intermediate