34 research outputs found

    Engineering of carbohydrate oxidoreductases for sensors and bio-fuelcells

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    Pyranose dehydrogenase (PDH) and pyranose 2-oxidase (POx) are flavoproteins that catalyze the oxidation of free, non-phosphorylated sugars to the corresponding ketosugars. Pyranose dehydrogenase has a broad substrate specificity for monosaccharides (and few disaccharides), but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen, whereas pyranose 2-oxidase shows pronounced specificity for glucose and displays high oxidase as well as dehydrogenase activity. For bio-fuelcell and sensor applications, oxygen reactivity is undesirable as it leads to electron leakage and the formation of damaging hydrogen peroxide; for biocatalytic applications, oxygen reactivity is advantageous, as oxygen is freely available and obviates downstream removal of undesired electron acceptors. Site-saturation mutagenesis libraries of eleven (POx) and twelve (PDH) residues around the active sites were screened for oxidase and dehydrogenase activities. In POx, variants T166R, Q448H, L545C, L547R and N593C displayed reduced oxidase activities (between 40% and 0.2% of the wildtype) concomitant with unaffected or even increased dehydrogenase activity, dependent on the electron acceptor used (DCPIP, 1,4-benzoquinone or ferricenium ion). Kinetic characterization showed that both affinity and turnover numbers can be affected. The switch from oxidase to dehydrogenase activity was also observed electrochemically using screen-printed electrodes. In this miniaturized set-up with a reaction volume of only 50 µL the dehydrogenase activity of variant N593C was entirely preserved in the presence of a suitable mediator, shuttling electrons from the FAD cofactor to the electrode surface. The oxidase activity, utilizing molecular oxygen as acceptor, is abolished in this variant. Of all variants of PDH that were produced by saturation mutagenesis, only variants of one position displayed increased oxygen reactivity to a minor degree. Histidine 103, carrying the covalently attached FAD cofactor, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y displayed a five-fold increase of oxygen reactivity. Stopped flow analysis revealed that the mutation slowed down the reductive half-reaction whereas the oxidative half-reaction was affected to a minor degree. No alterations in the secondary structure were observed, but disruption of the FAD bond had negative effects on thermal and conformational stability. We also engineered PDH by systematically removing several N-glycosylation sites, in order to improve performance by reducing the distance of the active site to the electrode surface, improving accessibility for redox polymers and facilitate denser enzyme packing on the electrode. One glycosylation site, N319, was found to be indispensable for functional expression and folding of the enzyme, as no active variants could be obtained. A variant with two sites, N75 and N175 near the active site entrance, exchanged against G and Q, respectively, showed significantly improved properties when used on electrodes with Osmium-based redox polymers (Mediated Electron Transfer) and a low level of Direct Electron Transfer. The lack of two glycosylation sites results in minor negative effects on expression yield and stability. Removal of a third site, N252, on the opposite side of the active site entrance, does not bring further improvements in catalysis and electron transfer, but is detrimental to functional expression and stability. The bulk of hyperglycosylation of the recombinantly expressed enzyme (observed in both Pichia pastoris and Saccharomyces cerevisiae) is located only on this one glycosylation site. Please click Additional Files below to see the full abstract

    RPE65 of Retinal Pigment Epithelium, A Putative Receptor Molecule for Plasma Retinol-Binding Protein, is Expressed in Human Keratinocytes

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    Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid–retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes

    Engineered Pyranose 2-Oxidase: Efficiently Turning Sugars into Electrical Energy

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    Due to the recent interest in enzymatic biofuel cells (BECs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale-devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi-rational protein design to improve the catalytic activity of the enzyme pyranose 2-oxidase (P2Ox) with its alternative soluble electron acceptors 1,4-benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BECs. Using a screening assay based on 96-well plates, we identified the variant H450G, which showed lower K-M and higher k(cat) values for both 1,4-benzoquinone and ferricenium ion compared to the wild-type enzyme, when either D-glucose or D-galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BECs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either "wired" to an osmium redox polymer or using soluble 1,4-benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4-fold compared to systems employing the wild-type enzyme. Our results show that by increasing the catalytic activity of the redox enzyme P2Ox with its alternative electron acceptors 1,4-benzoquinone and ferricenium ion, it is possible to achieve a higher energy output of an enzymatic BFC when using the same concentration of sugar substrate

    Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover.

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    The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme

    Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity

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    <div><p>Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH<sub>2</sub> and reoxidized in the second half-reaction by reducing molecular oxygen to H<sub>2</sub>O<sub>2</sub>. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H<sub>2</sub>O<sub>2</sub> production as well as reduced electron “leakage” to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties.</p></div

    Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.

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    <p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

    Identification of the exact site of oxidation in the peptide <sup>452</sup>DAFSYGAVQQSIDSR<sup>466</sup> by LC-ESI-MS/MS.

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    <p>The nearly complete series of C-terminal y-ions in the MS/MS spectrum of the double-charged ion of 830.4 confirms the peptide sequence. The +16 Da mass shift found for the ion y<sub>13</sub> further indicates that Phe454 is oxidised during substrate turnover and by H<sub>2</sub>O<sub>2</sub> treatment.</p

    Accessibility of methionine residues.

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    <p>Surface of the <i>T</i>. <i>multicolor</i> POx monomer in the vicinity of (<b>A</b>) Met417, showing the surface-exposed sulphur atom which is oxidised by H<sub>2</sub>O<sub>2</sub>, and (<b>B</b>) Met380 with its sulphur-containing side chain pointing towards the interior of the polypeptide matrix, in which it is buried and hence is not accessible from the surface.</p

    Mass spectrometric identification of methionine residues oxidized by H<sub>2</sub>O<sub>2</sub> during <i>Tm</i>POx inactivation.

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    <p>MALDI MS spectra were measured for unaffected POx (<b>A</b>), for POx inactivated during D-glucose oxidation (<b>B</b>) or for POx inactivated by endogenous H<sub>2</sub>O<sub>2</sub> (<b>C</b>). The selected MALDI spectra in the left panel illustrate that Met497 of the tryptic peptide ITDAYNMPQPTFDFR with a theoretical MH<sup>+</sup> of 1815.8 was extensively oxidised in <i>Tm</i>POx inactivated either during substrate turnover (<b>B</b>, left panel) or by H<sub>2</sub>O<sub>2</sub> treatment (<b>C</b>, left panel). In contrast, some methionine residues were found not to be oxidised during <i>Tm</i>POx inactivation as shown for Met74 of the peptide VAMFDIGEIDSGLK having a MH<sup>+</sup> of 1494.8 (right panel). The small signals at m/z 1510.8 are related to the oxidized form of the peptide generated due to the presence of air oxygen.</p
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