Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.     

Induction of lipid peroxidation in the erythrocytes during cholesterol oxidation catalysed by cholesterol oxidase

Using the chemiluminescence technique to assay the activity of cholesterol oxidase it has been shown that enzymic oxidation of cholesterol to cholest-4-en-3-one red cell membranes is accompanied by accumulation of lipid peroxidation products--malonyl dialdehyde (MDA). The amount of MDA formed was dependent on the amount of cholesterol oxidized. The free radical scavenger 4-methyl-2,6-ditretbutylphenol, the transition metal chelator EDTA and catalase inhibited lipid peroxidation in red blood cells. The participation of OH radicals in the initiation of lipid peroxidation in red cell membranes in the course of cholesterol oxidation is discussed.     

Cholesterol oxidation using hollow fiber dialyzer immobilized with cholesterol oxidase: preparation and properties

The surface of polyacrylonitrile hollow fibers were hydrolyzed and covalently bonded with cholesterol oxidase (COD) via glutaraldehyde. The immobilized amount of the COD increased with the concentration of glutaraldehyde. However, COD immobilized with 10% glutaraldehyde had higher activity than with other concentrations. The stabilities of immobilized COD to pH and temperature were higher than those of native enzyme. The immobilized enzyme retained 80% of initial activity after 15 days when stored at 4 degrees C, which was longer than native COD. After being reused six times, the COD-immobilized hollow fiber retained more than 80% of the activity.     

False-negative oxidase reaction as a result of medium acidification

Acidification of the culture medium may lead to a false-negative result of the oxidase reaction. Hence, if the medium contained a carbon source or another component that is convertible to acid, a negative oxidase reaction should be considered inconclusive. In order to eliminate false-negative reactions, a reagent freshly adjusted to pH 5–7 might be used; this reagent should be checked on known oxidase-negative strains. However, we recommend that the oxidase test be performed with the normal reagent after (sub)culturing on a non-acidogenic medium.     

Photophysical properties of Newkome-type dendrimers in aqueous medium.

Newkome-type first, second and third generation dendrimers, having t-butyl (GB), ethyl (GE) and carboxylic (GA) end groups, were synthesized. A pyrene group, which can act as fluorescent sensor, was attached to the core of the dendrimers and their photophysical properties in aqueous solution were studied. These dendrimers were found to aggregate in aqueous solution, which manifested as an excimer peak in the pyrene emission spectra for the first and second generation dendrimers with ethyl and t-butyl end groups. The excimer peak however was not seen in case of the third generation dendrimer. Dendrimers with carboxylic end groups, did not show the excimer peak in water, which implies the hydrophobic nature of the aggregation. It is observed that the intensity of the excimer peak decreases with the increase in the size of the dendrimer. Lifetime studies carried out on the first and second generation dendrimers showed the formation of excimer species as a risetime in the decay curve. The aggregation of the third generation dendrimer was proposed from the quenching studies using silver ions and CCl(4) as quenchers.     

Growth and cholesterol oxidation by Mycobacterium species in Tween 80 medium.

Mycobacterium strain DP was isolated from marine coastal sediment and tested for its ability to oxidize cholesterol in Tween 80-cholesterol (2.59 mM) medium. Strain DP degraded cholesterol to 4-cholesten-3-one (cholestenone), 4-androsten-3,17-dione (AD), 1,4-androstadien-3,17-dione (ADD), testosterone, and 1-dehydrotestosterone (DHT). Cholesterol disappeared in about 4 days. Cholestenone, AD, testosterone, and DHT accumulations were transient with peak concentrations of 300, 600, 30 to 40, and 21 microM. ADD production peaked after 6 days with a concentration of 1,100 microM. Peak ADD concentrations and production rates compared well with those reported for strain NRRL B3683 on cyclodextrin medium. Tween 80 medium was superior to finely dispersed cholesterol particles for both strains. In comparison, NRRL B3683 (patented for its ability to accumulate AD and ADD) on Tween 80 medium transiently accumulated more AD (approximately 1,000 microM) than did strain DP, but ADD accumulations (200 microM) were significantly lower than those for strain DP. Strain DP could be adapted to grow on ADD, which was initially inhibitory at 3.25 mM. ADD-adapted strain DP cultures produced approximately four times as much DHT from ADD than unadapted cultures did from cholesterol, showing that additional manipulation might enhance testosterone production. We believe that ADD toxicity might account for the low ADD accumulations by NRRL B3683 in Tween 80 medium.     

The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.     

Study of oxytetracycline solubility in an aqueous medium]

Solubility of oxytetracycline dihydrate in aqueous media was studied. It was shown that solubility of the drug in bidistillate at a temperature of 20 degrees was 195 gamma/ml, which was much lower than the requirements of some pharmacopoeia with respect to the drug solubility. Dependence of oxytetracycline dihydrate solubility in the aqueous medium on the values of pH and temperature was found. Indigency of the procedure described in the literature for determination of antibiotic solubility according to the dry weight of the filtrate on addition of large excesses of the solid phase to the system was shown.     

On the role of histidine 351 in the reaction of alcohol oxidation catalyzed by choline oxidase

Choline oxidase catalyzes the four-electron, flavin-linked oxidation of choline to glycine betaine with transient formation of an enzyme-bound aldehyde intermediate. The recent determination of the crystal structure of choline oxidase to a resolution of 1.86 A established the presence of two histidine residues in the active site, which may participate in catalysis. His466 was the subject of a previous study [Ghanem, M., and Gadda, G. (2005) Biochemistry 44, 893-904]. In this study, His351 was replaced with alanine using site-directed mutagenesis, and the resulting mutant enzyme was purified and characterized in its mechanistic properties. The results presented establish that His351 contributes to substrate binding and positioning and stabilizes the transition state for the hydride transfer reaction to the flavin, as suggested by anaerobic substrate reduction stopped-flow data. Furthermore, His351 contributes to the overall polarity of the active site by modulating the p K a of the group that deprotonates choline to the alkoxide species, as indicated by pH profiles of the steady-state kinetic parameters with the substrate or a competitive inhibitor. Surprisingly, His351 is not involved in the activation of the reduced flavin for reaction with oxygen. The latter observation, along with previous mutagenesis data on His466, allow us to conclude that choline oxidase must necessarily utilize a strategy for oxygen reduction different from that established for glucose oxidase, where other authors showed that the catalytic effect almost entirely arises from a protonated histidine residue.     

Influence of the reaction medium on the product distribution of peroxidase-catalysed oxidation of p-cresol

Summary p-Cresol was oxidized by hydrogen peroxide in a reaction catalysed by horseradish peroxidase and the low molecular weight products were investigated. In aqueous media Pummerer's ketone (I) was the dominating product but in organic media the product distribution was quite different; 2,2'-dihydroxy-5,5'-dimethyldiphenyl (II) was the main low molecular weight product. Similar product distributions were obtained with peroxidase adsorbed on a solid support and suspended in toluene and with peroxidase solubilized in a microemulsion containing the same solvent. The best selectivity for the formation of (II) was obtained when the enzyme was adsorbed on Celite and suspended in water-saturated chloroform with 0.5% (v/v) extra water added. The yield of low molecular weight products in this case was 28%; of this fraction, 95% was (II). Offprint requests to: P. Adlercreutz     

Cholesterol oxidase from Bordetella species promotes irreversible cell apoptosis in lung adenocarcinoma by cholesterol oxidation

Cholesterol oxidase (COD), an enzyme catalyzing the oxidation of cholesterol, has been applied to track the distribution of membrane cholesterol. Little investigations about the effect of COD on tumor cells have been performed. In the present study, we provided evidence that COD from Bordetella species (COD-B), induced apoptosis of lung cancer cells in vitro and in vivo. COD-B treatment inhibited Akt and ERK1/2 phosphorylation in dose- and time-dependent manner, which was not reversed and was even aggravated by cholesterol addition. Further investigation indicated that COD-B treatment promoted the generation of reactive oxygen species (ROS) and that cholesterol addition further elevated ROS levels. Moreover, COD-B treatment resulted in JNK and p38 phosphorylation, downregulation of Bcl-2, upregulation of Bax, activated caspase-3 and cytochrome C release, which likely responded to freshly produced hydrogen peroxide that accompanied cholesterol oxidation. Catalase pretreatment could only partially prevent COD-B-induced events, suggesting that catalase inhibited H₂O₂-induced signal transduction but had little effect on signal pathways involved in cholesterol depletion. Our results demonstrated that COD-B led to irreversible cell apoptosis by decreasing cholesterol content and increasing ROS level. In addition, COD-B may be a promising candidate for a novel anti-tumor therapy.Cholesterol is an essential component of the plasma membrane in eukaryotic cells and has an important role in maintaining structure integrity, receptor function, dynamics and ion channels in the plasma membrane.^{1, 2, 3} Cholesterol is a critical constituent for the formation of lipid rafts.⁴ Lipid rafts are plasma membrane microdomains locating abundant signaling molecules, such as caveolin-1 protein and epidermal growth factor receptor (EGFR).⁵ These molecules conduct a series of cellular functions, including cell proliferation and apoptosis.⁶ Because cholesterol has a bridging role in liquid-ordered rafts by binding tightly to the sphingolipids with saturated hydrocarbon chains, modification or depletion of membrane cholesterol is speculated to perturb the properties of lipid rafts.^{7, 8} Several studies have demonstrated that the depletion of membrane cholesterol led to the disruption of lipid rafts and dissociation of signaling molecules from lipid rafts, which generated aberrant signal transductions and disturbed cellular functions.⁹ Therefore, membrane cholesterol concentration is accurately regulated.¹⁰Cholesterol metabolism is disorganized in various tumors, such as prostate, lung, acute myeloid leukemia and breast cancer and especially in chemoresistant tumors.^{11, 12, 13, 14, 15} Solid tumors accumulate more cholesterol compared with normal tissue, which contributes to the proliferation, differentiation and migration of tumor cell.^{16, 17} The elevated content of membrane cholesterol modulates the activation of cellular surface receptors, such as EGFR.^{18, 19} Several reports have demonstrated that EGFR is upregulated in most malignant cells and stimulates the proliferation of cells by promoting the downstream activation of protein kinase B (Akt).^{20, 21, 22} Depletion of cholesterol from plasma membrane induces Akt inactivation and cell death.^{23, 24} Furthermore, the alteration of membrane cholesterol also affects the expression of the B-cell lymphoma/leukemia-2 (BCL-2) family members.^{21, 25} Therefore, it has been proposed that membrane cholesterol could potentially be a therapeutic target for tumors.The modification of membrane cholesterol can be mainly performed by methyl-beta-cyclodextrin (MβCD), statin and filipin.^{9, 21} Previous reports have indicated that MβCD could take up cholesterol into the internal hydrophobic pocket to form a MβCD–cholesterol complex and that the hydrophilic surface promoted the complex to dissolve in the aqueous phase.²⁶ Statins serve as competitive inhibitors to 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA) to block synthesis of cholesterol. Filipin, as a macrolide polyene antibiotic, binds specifically to cholesterol and causes the disruption of lipid rafts.²⁷ A common factor shared by the above three agents is that the cholesterol alteration caused by each of them can be reversed by the supplement of exogenous cholesterol. In view of high cholesterol in the modern diet, the effect of MβCD, statin and filipin on tumors would be attenuated, although prolonged suppression of cholesterol synthesis reduces the risk of advanced prostate cancer.²⁸Cholesterol oxidase (COD) is a flavoprotein from microorganisms that catalyzes the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide.²⁹ COD can convert membrane cholesterol to 4-cholesten-3-one and can inhibit the formation of lipid rafts.³⁰ Different from other cholesterol-depleting agents, COD disrupts lipid rafts by displacing cholesterol with 4-cholesten-3-one. However, because the action of COD is dependent on the microenvironment of membrane cholesterol, such as the phospholipid composition, cholesterol content and ionic strength, COD does not attack all cell types. So far, despite the fact that COD has been well known as a lipid raft destroyer, few investigations have been conducted to directly determine the impact of COD on signal transduction in cancer.In this study, we investigated whether COD purified from Bordetella species (COD-B) could suppress the growth of a lung adenocarcinoma cell line by its effect on membrane cholesterol. We provide the evidence that COD-B induces apoptosis in lung adenocarcinoma cells by catalyzing oxidation of membrane cholesterol and elevating reactive oxygen species (ROS) levels. Moreover, COD-B-induced apoptosis is not reversed and is aggravated by cholesterol supplementation.     

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.