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Electrochemical Characterization of the Pyranose 2-Oxidase Variant N593C Shows a Complete Loss Cite This: Phys

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Electrochemical Characterization of the Pyranose 2-Oxidase Variant N593C Shows a Complete Loss Cite This: Phys PCCP View Article Online PAPER View Journal | View Issue Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss Cite this: Phys. Chem. Chem. Phys., 2016, 18,32072 of the oxidase function with full preservation of substrate (dehydrogenase) activity† ab ab a ab Dagmar Brugger, Leander Su¨tzl, Kawah Zahma, Dietmar Haltrich, Clemens K. Peterbauerab and Leonard Stoica*a This study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen Creative Commons Attribution-NonCommercial 3.0 Unported Licence. reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was Received 31st August 2016, immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold- Accepted 23rd October 2016 nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for DOI: 10.1039/c6cp06009a utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active www.rsc.org/pccp equivalent dehydrogenase via a single residue exchange. This article is licensed under a Introduction member of the glucose–methanol–choline (GMC) oxidoreductase family,5,6 with obvious structural similarities. However, TmPOx The flavoprotein pyranose 2-oxidase (POx; pyranose: oxygen showsanaffinitytowardsD-glucose that is 72 times higher than 3 Open Access Article. Published on 28 October 2016. Downloaded 10/5/2021 8:38:43 AM. 2-oxidoreductase; synonym: glucose 2-oxidase; EC 1.1.3.10) that of AnGOx (Km values of 3.1 mM and 225 mM, respectively), from Trametes multicolor (TmPOx; synonym, Trametes ochracea)1 which complies perfectly with the concentration range of clinical was previously tested for glucose detection by immobilization interest. Also, the enzymatic efficiency (vmax/Km) of POx towards 1,2 À1 within an Os-hydrogel film as electron-relay. A major drawback D-glucose (9 U mM Â mg) is an order of magnitude higher than of using POx for glucose sensing, however, is its high affinity that of AnGOx (0.75 U mMÀ1 Â mg), thus at least similar current 3 towards oxygen, as determined by Decamps. POx has a Km value densities from a POx biosensor are expected. In contrast to GOx, towards oxygen of 0.46 mM, whereas the affinity of GOx from which is anomerically restricted to catalyze only the oxidation 7 3 Aspergillus niger (AnGOx) for oxygen is 6-fold lower as expressed by of b-D-glucose, POx converts both a- and b-D-glucose. The FAD 6,8 the higher Michaelis constant Km of 2.9 mM. Oxygen reduction at cofactor is covalently bound to the polypeptide chain of POx, POx thus interferes with the overall sensitivity towards glucose for whereas the FAD cofactor of GOx is only tightly entrapped a POx-based biosensor operating at low potentials, excluding the within the apoenzyme, and GDH relies on a freely diffusing 9 approach of detecting the enzymatically produced H2O2 by oxida- PQQ cofactor. tion at potentials above +600 mV vs. Ag|AgCl. As shown in Scheme 1, homotetrameric TmPOx10 catalyzes Aside from this limitation, POx presents several advantages the regioselective oxidation of aldopyranoses (D-glucose being over other oxidoreductases more commonly used for glucose its favoured substrate) at position C-2 forming the corres- sensing such as GOx and GDH.4 Similar to GOx, POx is also a ponding 2-ketoaldoses or osones.11 This catalytic oxidation of a sugar substrate by POx does not involve proton exchange and thus does not lead to a pH-shift, as is the case with GOx, which a Food Biotechnology Laboratory, University of Natural Resources and Life Sciences results in the formation of gluconic acid through hydrolysis of Vienna, Muthgasse 11, 1190 Vienna, Austria. E-mail: [email protected] D b BioToP – The Doctoral Programme on Biomolecular Technology of Proteins, the primary oxidation product -glucono-1,5-lactone following 12 Muthgasse 18, 1190 Vienna, Austria C1-oxidation of b-D-glucose. During this first half-reaction, the † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp06009a FAD-cofactor of POx is reduced to FADH2 (eqn (1)). The following 32072 | Phys. Chem. Chem. Phys., 2016, 18, 32072--32077 This journal is © the Owner Societies 2016 View Article Online Paper PCCP Scheme 1 Schematic representation of immobilised POx homotetramer onto SPE electrode and the main electrochemical processes at the electrode, (1) oxidase function (Ox-F) and (2) dehydrogenase function (DH-F), under- going at various polarization potentials in presence of catalytic oxidation of glucose at POx. half-reaction involves the reoxidation of the FADH2 cofactor by Fig. 1 (left plot) Cyclic voltammograms recorded for wt-POx in various reducing molecular oxygen to H O (eqn (2)).13 Possible alter- 2 2 conditions: (A) in buffer; (B) in presence of 300 mM glucose; and (C) in native electron acceptors for POx are quinones (eqn (3)), certain presence of 300 mM glucose and 700 mM 1,4-benzoquinone (BQ). (right 10 radicals or chelated metal ions. Considering the immobilization plot) Preliminary evidences of direct electron transfer (DET): magnification of POx at polarized interfaces as depicted in Scheme 1, the of CVs plot in the low potential range shows an upper shift in the oxidative resulting products of the enzymatic reaction are quasi-selectively direction in the presence of glucose (CV-B) in comparison to CV-A recorded in buffer. oxidized at the electrode surface (see eqn (4) and (5)). FAD + aldopyranose - FADH2 + 2-keto-aldopyranose Creative Commons Attribution-NonCommercial 3.0 Unported Licence. (enzyme mediator) between the reduced FAD cofactor of POx (1) and the polarized carbon electrode. 14 FADH2 +O2 - FAD + H2O2 (2) In a previous biochemical study, site saturation mutagenesis and microtiter plate screening was used to identify POx variants FADH + benzoquinone - FAD + hydroquinone (3) 2 with reduced oxidase activity but with preserved dehydrogenase hydroquinone - benzoquinone + 2H+ +2eÀ (4) activity, among them being the variant N593C. As seen in the crystal structure of TmPOx, residue N593 is involved in the 2H O +2H+ +2eÀ - H O (5) 2 2 2 electron transfer between the sugar substrate and the C(4a) The recycling of hydroquinone between reduced FAD-POx and position of the flavin6 and is located directly in the active site of This article is licensed under a the carbon electrode at potentials higher than +0 mV leads to TmPOx. In this work, we demonstrate the electrochemical a catalytic current in cyclic voltammetry that is characteristic consequences of the replacement of asparagine (N) at position for mediated electron transfer (MET). MET thus reflects the 593 with cysteine (C), resulting in a complete shut-down of the Open Access Article. Published on 28 October 2016. Downloaded 10/5/2021 8:38:43 AM. successive cascade of reactions (eqn (1), (3) and (4)). Therefore, oxidase function of POx. as seen in Fig. 1, the current generated by MET (IDH) is directly proportional to the magnitude of the dehydrogenase function (DH-F) of POx at potentials around +400 mV. As shown in Materials and methods Scheme 1, the second alternative route of electrons towards the Chemicals electrode is the cascade of reactions eqn (1), (2) and (5), All chemicals were of the highest commercially available purity involving oxygen reduction to H2O2, which is further oxidized at the electrode (at gold particles) at applied potentials higher and purchased from Merck (Darmstadt, Germany) or Sigma than +450 mV, leading to a peak potential at +650 mV. In the Aldrich (St. Louis, MO), unless otherwise stated. Gold Nanoparticle absence of a mediator, the resulting peak current at +650 mV (110GNP) modified Screen-Printed Carbon Electrodes (SPCEs) (DRP-110GNP) were purchased from DropSense (Llanera, Spain). (denoted IOX in Fig. 1) is proportional to the H2O2 produced by the enzyme, and is therefore directly proportional to the All solutions were prepared using a 50 mM sodium phosphate oxidase function (Ox-F) of POx. In the presence of glucose, buffer (pH 6.5). oxygen and mediator, irrespective of the electron pathway, the peak current at +650 mV shall also reflect the total catalytic Generation of wild-type TmPOx and its mutant N593C current (TCC) induced by POx immobilized on the electrode The generation of mutant N593C (herein denoted POx-C) and surface. The TCC value is therefore directly dependent on the its counter-partners N593S and N593K, as well as the purification concentrations of all three components, substrate (glucose), of the enzyme variants were carried out repetitive and separate by oxygen and mediator. two co-authors (DB and LS) according to previous publications.15,16 The gold–carbon electrode surface of a screen-printed electrode After concentration and washing steps, both POx enzymes (wt-POx plays a dual role for the chemisorption of POx on gold-nanoparticles and POx-C) were stored at 8 1C. The activity assay, applied to via existing His-tags and for recycling of 1,4-benzoquinone wild-type POx (wt-POx) and the mutant N593C (POx-C) was This journal is © the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 32072--32077 | 32073 View Article Online PCCP Paper previously described.17 Protein concentration was determined Results and discussion by the Bradford assay18 using the BioRad Protein Assay Kit with bovine serum albumin as standard.
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