Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cholesterol Oxidase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1978

Oxidation of 3beta-hydroxyandrostenes by the 3beta-hydroxy-steroid oxidase (cholesterol oxidase) from Brevibacterium sterolicum prior to their analysis by gas-liquid chromatography-mass spectrometry.

The 3beta-hydroxysteroid oxidase from Brevibacterium sterolicum has been applied to the oxidation of a number of 3beta-hydroxyandrostenes, including polar steroids containing up to three other hydroxylic groups. The substrates, products, and derivatives thereof have been examined by gas-liquid chromatography. Retention index increments for these conversions, and for parallel transformations of other steroids, show considerable regularities, and together with mass spectrometric data afford characteristic structural information.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

A colorimetric assay for measuring cell-free and cell-bound cholesterol oxidase.

Cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) catalyzes the conversion of sterol delta 5-3 beta-alcohol to the corresponding delta 4-3-ketone with the reduction of oxygen to hydrogen peroxide. Rhodococcus species GK 1, a soil isolated microbe, produces an extracellular and a membrane-bound cholesterol oxidase; the latter is bound to the outer surface of the microbial cell membrane. A simple and sensitive assay is described to measure the two enzyme types; no enzyme extraction is needed for measuring the membrane-bound cholesterol oxidase. In this assay, hydrogen peroxide is reduced by the chromogen 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of horseradish peroxidase, and the increased absorbance is followed continuously at 600 nm (epsilon m = 1.82 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C). The standardized assay medium contained 46.9 mM sodium-potassium phosphate buffer pH 7.0, 0.16% Triton X-100, 312.5 microM ABTS, 50 micrograms peroxidase (12.5 units at 25 degrees C), 6.25% isopropanol, 306.3 microM cholesterol or other sterols (kept in solution with isopropanol), and cholesterol oxidase. Oxidation of one molecule of cholesterol by cholesterol oxidase gives one molecule of hydrogen peroxide which reacts with two molecules of ABTS. The method is reproducible and the results correlate well with those obtained by measuring the absorbance of delta 4-cholest-3-one at 240 nm (epsilon m = 1.40 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C) and by the method of Allain et al. (Clin. Chem. 20, 470-475, 1974).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Propanol↗
PubMed1982

Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase.

Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.

Aspergillus niger↗
PubMed2001

Dissection of a flavoenzyme active site: the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase is a member of the glucose-methanol-choline (GMC) oxidoreductase family that is characterized by a conserved topology. We review our investigations into the reactivity of the Streptomyces cholesterol oxidase cofactor, flavin adenine dinucleotide (FAD), and the role of active-site residues. All of our mutagenesis, enzyme inhibition, and kinetic data demonstrate that the cofactor catalyzes oxidation of alcohols to ketones, but not oxygenation of carbon. Cholesterol oxidase catalyzes two reactions, oxidation and isomerization, in one active site, presumably because of the susceptibility of the reaction intermediate cholest-5-en-3-one to radical oxidation. This bifunctionality is not a shared characteristic with other GMC oxidoreductase family members. Furthermore, we have characterized the unusual inactivation of FAD by electrophilic substitution at C6 of the isoalloxazine ring upon ring opening of a cyclopropyl steroid. Another member of the GMC oxidoreductase family, methanol oxidase, is also inactivated by a cyclopropanol suggesting that inhibition by cyclopropanol inhibitors may be diagnostic of membership in this family.

Alcohols↗
PubMed1993

[The effect of structural organization of cholesterol aggregates in aqueous-organic media on its oxidation reaction, catalyzed by cholesterol oxidase].

Cholesterol oxidation by cholesterol oxidase in homogeneous water-organic mixtures without the use of surface-active agents and the effects of reaction medium parameters on this process have been studied. It has been shown that the nature of the organic solvent, buffer salts, pH and the "age" of the cholesterol solutions determine the formation of the "substrate" properties of sterol molecules in the aggregates which is manifested as changes in the kinetic parameters of the oxidation reaction and in the spectrophotometric characteristics of the enzymatic oxidation product, 4-cholesten-3-one. Such changes are the most vividly pronounced during oxidation of "aged" cholesterol solutions prepared 24 hrs before the reaction and are concomitant with significant reductions in the maximal reaction rate, Km values of cholesterol oxidase for cholesterol and the effective molar extinction coefficient for 4-cholesten-3-one. Sonication of "aged" cholesterol solutions partly restores these indices. The observed phenomena seem to be due to the different extent of aggregation of cholesterol molecules in solutions. Other factors influencing the value of the molar extinction coefficient for 4-cholesten-3-one, such as hypochromicity and stray light, are also discussed. The enzyme has a broad pH optimum at pH 7.0. The Michaelis constant for cholesterol does not appreciably change in the pH range studied (6.0-8.0) and constitutes approximately 15 microM for freshly prepared cholesterol solutions containing 10% propanol-1. The catalytic constant measured under the same conditions is about 22 s-1 at 20 degrees C and pH 7.0. The stability of cholesterol solutions is strongly influenced by the pH of the reaction medium, nature of the organic solvent and buffer salts. Weakly alkaline solutions prove to be the most stable ones. Regardless of the nature of the buffer salt its concentration in the range of 0.02-0.2 M does not affect the reaction.

Buffers↗
PubMed1993

Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases.

Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3 beta-hydroxy steroids with a double bond at delta 5-delta 6 of the steroid ring backbone. The crystal structure of the free enzyme in the absence of a steroid substrate has previously been determined. In this paper we report the crystal structure of the complex of cholesterol oxidase with the steroid substrate dehydroisoandrosterone, refined at 1.8-A resolution. The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-A resolution. The steroid is buried within the protein in an internal cavity which, in the free enzyme crystal structure, was occupied by a lattice of water molecules. The conformations of a number of side chains lining the active-site cavity have changed in order to accommodate the steroid substrate. A loop region of the structure between residues 70 and 90 differs significantly between the substrate-free and substrate-bound forms of the enzyme, presumably to facilitate binding of the steroid. The hydroxyl group of the steroid substrate is hydrogen-bonded to both the flavin ring system of the FAD cofactor and a bound water molecule. FAD-dependent cholesterol oxidase shares significant structural homology with another flavoenzyme, glucose oxidase, suggesting that it might also be a member of the glucose-methanol-choline (GMC) oxidoreductase family. Although there is only limited sequence homology, a superposition of these two structures reveals a conserved histidine residue within hydrogen-bonding distance of the active-site water molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Brevibacterium↗
PubMed2006

Structural and kinetic analyses of the H121A mutant of cholesterol oxidase.

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.

Amino Acid Substitution↗
PubMed1996

Sterol peroxidation by Pseudomonas fluorescens cholesterol oxidase.

Cholesterol is oxidized by commercially available Pseudomonas fluorescens cholesterol oxidase to 6 beta-hydroperoxycholest-4-en-3-one as the initial product, with none of the expected produce, cholest-4-en-3-one, formed. The transformation indicates that P. fluorescens cholesterol oxidase also acts as a flavoprotein dioxygenase.

Cholesterol↗
PubMed2001

The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.

Asparagine↗
PubMed1998

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.

Asparagine↗
PubMed1976

Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase.

Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods.

Carboxylic Ester Hydrolases↗
PubMed1998

The importance of GLU361 position in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase stereospecifically isomerizes cholest-5-en-3-one to cholest-4-en-3-one. When the base catalyst for isomerization, Glu361, is mutated to Asp, the rate of deprotonation of cholest-5-en-3-one is not affected, but protonation of the dienolic intermediate becomes rate-limiting. This may be a consequence of the large distance between the catalytic base and carbon-6 of the intermediate in the mutant enzyme.

Binding Sites↗
PubMed1977

The reactivity of human erythrocyte membrane cholesterol with a cholesterol oxidase.

Cholesterol oxidase (EC 1.1.3.6, Brevibacterium sp.), which catalyzes the reaction: cholesterol + O2 leads to delta4-cholestenone + H2O2, has no effect on the cholesterol of intact (human) erythrocytes and of "resealed" ghosts, when it is present only outside these ghosts. The cholesterol of "leaky" ghosts, of "resealed" ghosts with enzyme trapped within, and of "inside-out" vesicles, was completely oxidized. This pattern indicates that the inner ((cytoplasmic) membrane surface must be exposed to the enzyme for the reaction to occur, and that outer surface cholesterol only becomes reactive after the membrane has been degraded by the oxidation of inner surface cholesterol. The enzymatic oxidations followed monotonic first-order kinetics, and hence gave no evidence to support the two states of cholesterol in the membrane that had been postulated earlier from studies on the plasma lipoprotein extraction of cholesterol from the membrane.

Brevibacterium↗
PubMed2003

Comparison of biosensors based on entrapment of cholesterol oxidase and cholesterol esterase in electropolymerized films of polypyrrole and diaminonaphthalene derivatives for amperometric determination of cholesterol.

Cholesterol amperometric biosensors constructed with enzymes entrapped in electropolymerized layers of polypyrrole and poly-naphthalene derivative polymers are compared. The biosensors are based on entrapment of cholesterol oxidase and/or cholesterol esterase in monolayer or multilayer films electrochemically synthesised from pyrrole, 1,8-diaminonaphthalene (1,8-DAN), and 1,5-diaminonaphthalene (1,5-DAN) monomers. Seven configurations were assayed and compared, and different analytical properties were obtained depending on the kind of polymer and the arrangement of the layers. The selectivity properties were evaluated for the different monolayer and bilayer configurations proposed as a function of the film permeation factor. All the steps involved in the preparation of the biosensors and determination of cholesterol were carried out in a flow system. Sensitivity and selectivity depend greatly on hydrophobicity, permeability, compactness, thickness, and the kind of the polymer used. In some cases a protective outer layer of non-conducting poly( o-phenylenediamine) polymer improves the analytical characteristics of the biosensor. A comparative study was made of the analytical performance of each of the configurations developed. The biosensors were also applied to the flow-injection determination of cholesterol in a synthetic serum.

Biosensing Techniques↗
PubMed1999

Amperometric determination of total cholesterol at gold electrodes covalently modified with cholesterol oxidase and cholesterol esterase with use of thionin as an electron mediator.

Immobilization of cholesterol oxidase (EC 1.1.3.6) (ChOx) on a gold electrode was attempted by cross-linking using glutaraldehyde between ChOx molecules and a self-assembled monolayer of 2-aminoethanethiolate. The resulting electrode (ChOx/Au) exhibits an amperometric response to free cholesterol in the presence of thionin as an electron mediator, and a steady-state response is obtained approximately 60 s after injection of cholesterol into the electrolyte solution. Coimmobilization of cholesterol esterase (EC 3.1.1.13) (ChE) and ChOx (ChE/ChOx/Au) allows the amperometric determination of both esterified cholesterol and free cholesterol. Cyclic voltammetry of the ChE/ChOx/Au and the dependence of the amperometric response to cholesterol on the concentration of thionin suggest that thionin is encapsulated in the enzyme film on the electrode surface. Apparent Michaelis constants of the ChOx/Au and the ChE/ChOx/Au electrodes suggest that the amperometric response was controlled by penetration of the reaction substrate into the films of the enzyme(s). The concentration of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, is in good agreement with that determined by the well-established technique using colorimetry.

Cholesterol↗
PubMed2004

Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method.

BACKGROUND: Cholesterol oxidase is used for the determination of serum cholesterol. It can be derived from Streptomyces, Pseudomonas fluorescens, Cellulomonas, and Brevibacterium. This study compared the performance characteristics of four enzymes in the endpoint cholesterol determination. METHODS: Using the Mega analyzer, we studied assay optimization, linearity, precision, recovery, interference, stability, and compared 110 patient samples. RESULTS: The linearity for the four enzymes was up to 13.0 mmol/l at the optimal enzyme activity. The average within-run CVs ranged from 1.6% to 1.9% and between-day ranged from 2.8% to 3.0%, within the NCEP analytical criteria. The analytical recoveries obtained from four reagents ( approximately 96.5%) were excellent. The assays using these enzyme sources compared favorably with the commercial method and appeared accurate near the clinical decision cut-points. Hemoglobin concentration at 1.9 g/l interfered with the P. fluorescens cholesterol oxidase. Bilirubin caused a negative interference while lipemia generated a positive interference with all enzyme sources. Reagents were stable up to 6 weeks. CONCLUSIONS: Streptomyces, Cellulomonas, and Brevibacterium were essentially analytically equivalent. Streptomyces and Cellulomonas cholesterol oxidase are one-quarter as expensive Brevibacterium. Cellulomonas is a new source of cholesterol oxidase for determining serum cholesterol by the endpoint method.

Bilirubin↗
PubMed1992

Cholesterol oxidase susceptibility of cholesterol and 5-androsten-3 beta-ol in pure sterol monolayers and in mixed monolayers containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

This study has examined the importance of the isocaproic side chain at C-17 of cholesterol to sterol/phospholipid interactions in monolayer membranes and to the cholesterol oxidase-susceptibility of cholesterol in pure and mixed monolayers at the air/water interface. The interactions between cholesterol or 5-androsten-3 beta-ol (which lacks the C-17 side chain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in monolayers indicated that 5-androsten-3 beta-ol was not very efficient in causing condensation of the monolayer packing of POPC. Whereas cholesterol condensed the packing of POPC at all molar fractions examined (i.e., 0.25, 0.50 and 0.75 with regard to POPC), 5-androsten-3 beta-ol caused a slight condensing effect on POPC packing only in the equimolar mixture. The mean molecular area requirement of 5-androsten-3 beta-ol (in pure sterol monolayers at different lateral surface pressures) was 2.2-6.7% less than that observed for cholesterol. The pure 5-androsten-3 beta-ol monolayer also collapsed at lower lateral surface pressures compared with the pure cholesterol monolayer (34 mN/m and 45 mN/m, respectively). The cholesterol oxidase (Streptomyces sp.) catalyzed oxidation of cholesterol or 5-androsten-3 beta-ol in pure monolayers in the air/water interface (10 mN/m) proceeded with very similar rates, indicating that the enzyme did not recognize that the C-17 side chain of 5-androsten-3 beta-ol was missing. The oxidation of cholesterol or 5-androsten-3 beta-ol in mixed POPC-containing monolayers (equimolar mixture) also revealed similar reaction rates, although the reaction was slower in the mixed monolayer compared with the pure sterol monolayer. When the oxidation of cholesterol and 5-androsten-3 beta-ol was examined by monitoring the production of H2O2 (the sterol was solubilized in 2-propanol and the assay conducted in phosphate buffer), the maximal reaction rate observed with 5-androsten-3 beta-ol was only about 41% of that measured with cholesterol. From the cholesterol oxidase point-of-view, it can be concluded that the enzyme did not recognize the C-17 side chain of cholesterol (or lack of it in 5-androsten-3 beta-ol), when the sterol was properly oriented as a monolayer at the air/water interface. However, when the substrate was presented to the enzyme in a less controlled orientation (organic solvent in water), 5-androsten-3 beta-ol may have oriented itself unfavorably compared with the orientation of cholesterol, thereby leading to slower oxidation rates.

1-Propanol↗