Homemade cofactors: self-processing in galactose oxidase.
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ABSTRACT:
SUBMITTER: Xie L
PROVIDER: S-EPMC60785 | biostudies-literature | 2001 Nov
REPOSITORIES: biostudies-literature
Publications
Homemade cofactors: self-processing in galactose oxidase.
Proceedings of the National Academy of Sciences of the United States of America 20011101 23
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Project description:Galactose oxidase (EC ) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa pro-sequence at the N terminus. Previous work has shown that the aerobic addition of Cu(2+) to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 A, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.
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Project description:A reagent-free colorimetric method for galactose quantification using a composite of cerium oxide nanoparticles (nanoceria) and galactose oxidase (Gal Ox) entrapped in an agarose gel was developed. In the presence of galactose, the Gal Ox entrapped within the agarose gel catalyzed the oxidation of galactose to generate H2O2, which induced a color change from white to intense yellow. This reaction occurred without any chromogenic substrate. This color transition is presumed to be due to the H2O2-mediated alteration of the oxidation state of cerium ions present on the surface of the nanoceria. The intensity of color change was quantified by acquiring an image with a conventional smartphone, converting the image to cyan-magenta-yellow-black (CMYK) mode, and subsequently analyzing the image using the ImageJ software. Using this strategy, galactose concentration was specifically determined with excellent sensitivity of as low as 0.05 mM. The analytical utility of the assay was successfully verified by correctly determining diverse levels of galactose in human serum, which is enough to diagnose galactosemia, a genetic disorder characterized by the malfunctioning of enzymes responsible for galactose metabolism. The assay employing a hydrogel composite with entrapped nanoceria and Gal Ox, is a simple, cost-effective, and rapid colorimetric assay for galactose quantification, without using any chromogenic reagent. This cost-effective method has great potential for the diagnosis of galactosemia and is highly promising in comparison to the laborious instrumentation-based methods currently in use.
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Project description:Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure-function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications.
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Project description:Colorimetric assays are a rapid, scalable technique well suited to enzyme activity screening. However, side reactions or chromogenic reagent instability can result in false positives or false negatives that compromise the accuracy of such assays. Here, we identify three classes of compounds incompatible with the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) colorimetric assay for galactose oxidase activity. Dark green ABTS·+ cationic radicals indicating enzyme activity can get quenched to yield colorless solutions or couple with substrates to form differently colored adducts, thus preventing accurate colorimetric measurements. These side reactions limit the utility of the ABTS assay and introduce uncertainty in the substrate scope to which it is applicable. We have investigated the underlying mechanisms behind these side reactions to conclude that free radical scavengers, phenols with electron-donating substituents, and β,γ-unsaturated aryl ketones are incompatible with the ABTS colorimetric assay. In search of a viable alternative, we developed an assay using 2,4-dinitrophenylhydrazine under neutral conditions with isopropyl alcohol as a solubilizing agent. The use of neutral conditions was found to be critical to avoid hydrolysis of hydrazone adducts, ensuring reproducible measurements. Our assay is compatible with free radical scavengers (R2 = 0.98), phenols with electron-donating substituents (R2 = 0.97), and β,γ-unsaturated aryl ketones (R2 = 0.88). This modified assay enables galactose oxidase activity screening across a broader substrate scope, thus facilitating enzyme use for more practical applications.
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Project description:A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His2 -Tyr2 (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see-saw-type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one-electron oxidation that is centered at the phenolate donor leading to a copper-coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.
| S-EPMC6857004 | biostudies-literature
Project description:Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.
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Project description:The use of enzymes as catalysts in chemical synthesis offers advantages in terms of clean and highly selective transformations. Galactose oxidase (GalOx) is a remarkable enzyme with several applications in industrial conversions as it catalyzes the oxidation of primary alcohols. We have investigated the wiring of GalOx with a redox polymer; this enables mediated electron transfer with the electrode surface for its potential application in biotechnological conversions. As a result of electrochemical regeneration of the catalytic center, the formation of harmful H2 O2 is minimized during enzymatic catalysis. The introduced bioelectrode was applied to the conversion of bio-renewable platform materials, with glycerol as model substrate. The biocatalytic transformations of glycerol and 5-hydroxymethylfurfural (HMF) were investigated in a circular flow-through setup to assess the possibility of substrate over-oxidation, which is observed for glycerol oxidation but not during HMF conversion.
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Project description:Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 μM of LacOX (eq. 30 μg L-1). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs.
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Project description:Integrating sulfanyl substituents into copper-bonded phenoxyls significantly alters their optical and redox properties and provides insight into the influence of cysteine modification of the tyrosine cofactor in the enzyme galactose oxidase. The model complexes [1(SR2)](+) are class II mixed-valent Cu(II)-phenoxyl-phenolate species that exhibit intervalence charge transfer bands and intense visible sulfur-aryl π → π* transitions in the energy range, which provides a greater spectroscopic fidelity to oxidized galactose oxidase than non-sulfur-bearing analogs. The potentials for phenolate-based oxidations of the sulfanyl-substituted 1(SR2) are lower than the alkyl-substituted analogs by up to ca. 150 mV and decrease following the steric trend: -S(t)Bu > -S(i) Pr > -SMe. Density functional theory calculations suggest that reducing the steric demands of the sulfanyl substituent accommodates an in-plane conformation of the alkylsulfanyl group with the aromatic ring, which stabilizes the phenoxyl hole by ca. 8 kcal mol(-1) (1 kcal = 4.18 kJ; 350 mV) through delocalization onto the sulfur atom. Sulfur K-edge X-ray absorption spectroscopy clearly indicates a contribution of ca. 8-13% to the hole from the sulfur atoms in [1(SR2)](+). The electrochemical results for the model complexes corroborate the ca. 350 mV (density functional theory) contribution of hole delocalization on to the cysteine-tyrosine cross-link to the stability of the phenoxyl radical in the enzyme, while highlighting the importance of the in-plane conformation observed in all crystal structures of the enzyme.
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Project description:A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
| S-EPMC4370742 | biostudies-literature