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James W. Whittaker - One of the best experts on this subject based on the ideXlab platform.
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Radiation inactivation of Galactose Oxidase, a monomeric enzyme with a stable free radical
Protein science : a publication of the Protein Society, 2009Co-Authors: Ellis S. Kempner, James W. Whittaker, Jay H. MillerAbstract:To determine the radiation sensitivity of Galactose Oxidase, a 68 kDa monomeric enzyme containing a mononuclear copper ion coordinated with an unusually stable cysteinyl-tyrosine (Cys-Tyr) protein free radical. Both active enzyme and reversibly rendered inactive enzyme were irradiated in the frozen state with high-energy electrons. Surviving polypeptides and surviving enzyme activity were analyzed by radiation target theory giving the radiation sensitive mass for each property. In both active and inactive forms, protein monomer integrity was lost with a single radiation interaction anywhere in the polypeptide, but enzymatic activity was more resistant, yielding target sizes considerably smaller than that of the monomer. These results suggest that the structure of Galactose Oxidase must make its catalytic activity unusually robust, permitting the enzymatic properties to survive in molecules following cleavage of the polymer chain. Radiation target size for loss of monomers yielded the mass of monomers indicating a polypeptide chain cleavage after a radiation interaction anywhere in the monomer. Loss of enzymatic activity yielded a much smaller mass indicating a robust structure in which catalytic activity could be expressed in cleaved polypeptides.
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The radical chemistry of Galactose Oxidase.
Archives of biochemistry and biophysics, 2005Co-Authors: James W. WhittakerAbstract:Galactose Oxidase is a free radical metalloenzyme containing a novel metalloradical complex, comprised of a protein radical coordinated to a copper ion in the active site. The unusually stable protein radical is formed from the redox-active side chain of a cross-linked tyrosine residue (Tyr-Cys). Biochemical studies on Galactose Oxidase have revealed a new class of oxidation mechanisms based on this free radical coupled-copper catalytic motif, defining an emerging family of enzymes, the radical-copper Oxidases. Isotope kinetics and substrate reaction profiling have provided insight into the elementary steps of substrate oxidation in these enzymes, complementing structural studies on their active site. Galactose Oxidase is remarkable in the extent to which free radicals are involved in all aspects of the enzyme function: serving as a key feature of the active site structure, defining the characteristic reactivity of the complex, and directing the biogenesis of the Tyr-Cys cofactor during protein maturation.
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Stereoselective hydrogen abstraction by Galactose Oxidase.
Biochemistry, 2004Co-Authors: Stefan G. Minasian, Mei M. Whittaker, James W. WhittakerAbstract:The fungal enzyme Galactose Oxidase is a radical copper Oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on Galactose Oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-α-d-Galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the α,α‘-dideuterated alcohol to the corresponding 2H-labeled aldehyde, followed by asymmetric hydroboration using α-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by ...
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Cu(I)-dependent biogenesis of the Galactose Oxidase redox cofactor.
The Journal of biological chemistry, 2003Co-Authors: Mei M. Whittaker, James W. WhittakerAbstract:Galactose Oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing Galactose Oxidase from preGalactose Oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature Galactose Oxidase forms rapidly following exposure of a preGalactose Oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of preGalactose Oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the preGalactose Oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature Galactose Oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.
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expression of recombinant Galactose Oxidase by pichia pastoris
Protein Expression and Purification, 2000Co-Authors: Mei M. Whittaker, James W. WhittakerAbstract:Abstract Galactose Oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of Galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose Oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature Galactose Oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed Galactose Oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25°C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active Galactose Oxidase suitable for a wide range of biomedical and bioanalytical applications.
Christine Mousty - One of the best experts on this subject based on the ideXlab platform.
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Galactose Oxidase prussian blue based biosensors
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:This paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor.
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Galactose Oxidase/Prussian Blue Based Biosensors
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:International audienceThis paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor
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Galactose Oxidase/Prussian Blue Based Biosensors.
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:This paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor.
Mei M. Whittaker - One of the best experts on this subject based on the ideXlab platform.
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Stereoselective hydrogen abstraction by Galactose Oxidase.
Biochemistry, 2004Co-Authors: Stefan G. Minasian, Mei M. Whittaker, James W. WhittakerAbstract:The fungal enzyme Galactose Oxidase is a radical copper Oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on Galactose Oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-α-d-Galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the α,α‘-dideuterated alcohol to the corresponding 2H-labeled aldehyde, followed by asymmetric hydroboration using α-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by ...
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Cu(I)-dependent biogenesis of the Galactose Oxidase redox cofactor.
The Journal of biological chemistry, 2003Co-Authors: Mei M. Whittaker, James W. WhittakerAbstract:Galactose Oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing Galactose Oxidase from preGalactose Oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature Galactose Oxidase forms rapidly following exposure of a preGalactose Oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of preGalactose Oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the preGalactose Oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature Galactose Oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.
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expression of recombinant Galactose Oxidase by pichia pastoris
Protein Expression and Purification, 2000Co-Authors: Mei M. Whittaker, James W. WhittakerAbstract:Abstract Galactose Oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of Galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose Oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature Galactose Oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed Galactose Oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25°C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active Galactose Oxidase suitable for a wide range of biomedical and bioanalytical applications.
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Expression of recombinant Galactose Oxidase by Pichia pastoris.
Protein expression and purification, 2000Co-Authors: Mei M. Whittaker, James W. WhittakerAbstract:Galactose Oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of Galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose Oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature Galactose Oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed Galactose Oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active Galactose Oxidase suitable for a wide range of biomedical and bioanalytical applications.
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Synthesis, Structure, and Properties of a Model for Galactose Oxidase.
Inorganic chemistry, 1996Co-Authors: Mei M. Whittaker, Walter R. Duncan, James W. WhittakerAbstract:An active-site analog of the radical copper enzyme Galactose Oxidase has been prepared from a synthetic tripod chelate ((2-pyridylmethyl)[(2-hydroxy-3,5-dimethylphenyl)methyl][(2-hydroxy-5-methyl-3-(methylthio)phenyl)methyl]amine, duncamine (dnc)) that binds a single Cu(II) ion through phenolate, thioether-substituted phenolate, and pyridylamine arms. The Cu complex crystallizes as a dinucleated dimer bridged by phenolate oxygens, and the structure has been determined by X-ray crystallography. Addition of pyridine (or other coordinating bases) dissociates the complex into a monomeric derivative that has been characterized spectroscopically (optical absorption and EPR) and electrochemically. The model provides insight into the properties of a mutant form of Galactose Oxidase which retains the same copper ligand complement as the wild type protein but lacks catalytic activity.
Franck Charmantray - One of the best experts on this subject based on the ideXlab platform.
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Galactose Oxidase prussian blue based biosensors
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:This paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor.
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Galactose Oxidase/Prussian Blue Based Biosensors
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:International audienceThis paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor
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Galactose Oxidase/Prussian Blue Based Biosensors.
Electroanalysis, 2015Co-Authors: Franck Charmantray, Nadia Touisni, Laurence Hecquet, Thierry Noguer, Christine MoustyAbstract:This paper describes the development of an amperometric biosensor based on Galactose Oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor.
Nicholas J. Turner - One of the best experts on this subject based on the ideXlab platform.
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a single enzyme oxidative cascade via a dual functional Galactose Oxidase
ACS Catalysis, 2018Co-Authors: William R Birmingham, Nicholas J. TurnerAbstract:The Galactose Oxidase (GOase) M3-5 variant, previously engineered for enantioselective oxidation of (R)-secondary alcohols, is now shown to catalyze the sequential four-electron oxidation of substituted benzylic and heteroaromatic benzylic alcohols to the corresponding carboxylic acids via the intermediate aldehyde. Aldehyde oxidation has been shown to occur on the hydrated (gem-diol) form of the aldehyde, and hence the activity of this second oxidation step is primarily determined by the effects of substituents on the aromatic ring. The demonstration of GOase for “through oxidation” of alcohols to carboxylic acids represents a fusion of the activities of two distinct copper radical Oxidases (Galactose Oxidase and glyoxal Oxidase) into a single enzyme sequence with potential applications as an abbreviated oxidative cascade.
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A Single Enzyme Oxidative “Cascade” via a Dual-Functional Galactose Oxidase
2018Co-Authors: William R. Birmingham, Nicholas J. TurnerAbstract:The Galactose Oxidase (GOase) M3‑5 variant, previously engineered for enantioselective oxidation of (R)-secondary alcohols, is now shown to catalyze the sequential four-electron oxidation of substituted benzylic and heteroaromatic benzylic alcohols to the corresponding carboxylic acids via the intermediate aldehyde. Aldehyde oxidation has been shown to occur on the hydrated (gem-diol) form of the aldehyde, and hence the activity of this second oxidation step is primarily determined by the effects of substituents on the aromatic ring. The demonstration of GOase for “through oxidation” of alcohols to carboxylic acids represents a fusion of the activities of two distinct copper radical Oxidases (Galactose Oxidase and glyoxal Oxidase) into a single enzyme sequence with potential applications as an abbreviated oxidative cascade
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Heavily fluorinated carbohydrates as enzyme substrates: Oxidation of tetrafluorinated Galactose by Galactose Oxidase
Chemical Communications, 2011Co-Authors: Avgousta Ioannou, Roxana S. Timofte, Elena Cini, Nicholas J. Turner, Sabine L Flitsch, Bruno LinclauAbstract:Galactose Oxidase (GOase) was shown to oxidise several C2/C3 fluorinated Galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated Galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated sugar.
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Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution.
Journal of the American Chemical Society, 2011Co-Authors: Julie B. Rannes, Avgousta Ioannou, Sabine L Flitsch, Simon C. Willies, Gideon Grogan, Carsten Behrens, Nicholas J. TurnerAbstract:A directed evolution approach has been used for the generation of variants of Galactose Oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.
