Cholesterol heterogeneity in the plasma membrane of epithelial cells.

The distribution of cholesterol in the plasma membrane of epithelial cells has been determined using renal brush border vesicles as a model. In brush borders treated with Brevibacterium sp. or Nocardia erythropolis cholesterol oxidases, a significant fraction of the free cholesterol was oxidized rapidly, without glutaraldehyde fixation, and the remaining cholesterol was oxidized at a slower rate. The size of the readily accessible cholesterol pool, however, depended on the enzyme used, varying from 16% of the total in membranes treated with N. erythropolis oxidase, to 27% using the Brevibacterium sp. enzyme. The slowly accessible pool detected by the Brevibacterium oxidase was suppressed upon sphingomyelinase addition. On the other hand, the restricted activity of the Nocardia oxidase might depend on phosphatidylcholine/cholesterol interactions. These results indicate that cholesterol distribution is heterogeneous in intact renal brush border vesicles. They suggest that, as proposed for model system [Demel, R.A. Jansen, J.W.C.M., van Dijck, P.W.M., & van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 465, 1-10], preferential interactions between some classes of phospholipids and cholesterol define cholesterol pools in the plasma membrane of epithelial cells.     

Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.     

Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane

Cholesterol present in intact brush-border membrane vesicles made from rabbit small intestine is a poor substrate for cholesterol oxidase (EC 1.1.3.6, from Nocardia sp. and Nocardia erythropolis). It becomes susceptible to oxidation by the enzyme only after the addition of detergent, e.g., Triton X-100, in quantities sufficient to disrupt the membrane. This is also true for cholesterol present in bilayers of small unilamellar phosphatidylcholine or phosphatidylserine vesicles made by ultrasonication. The data presented here on intestinal brush-border membrane are in good agreement with results reported on other biological membranes, e.g., from erythrocytes and vesicular stomatitis virus, but are somewhat different from those on rat intestinal brush-border membrane. Our results on phospholipid bilayers agree well with published work on model membranes. From the work presented we conclude that, with our present understanding, cholesterol oxidase can hardly be used to probe the distribution of cholesterol in biological membranes. A prerequisite for using the enzyme successfully as such a probe would be the understanding of the factors controlling the interaction of the enzyme with its substrate cholesterol. The question under which conditions cholesterol oxidase could be useful for probing the distribution and preferred location of cholesterol in biological membranes is discussed.     

Circular dichroism studies on flavoproteins containing covalently bound coenzymes

Absorption and circular dichroism spectra of cholesterol oxidase from Schizophyllum commune and choline oxidase from Alcaligenes sp. were measured and compared. The prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD (1, 2), while that of choline oxidase is 8 alpha-[N(3)-histidyl]-FAD (3). In the CD spectra of the two enzymes in either the oxidized or reduced state, the corresponding bands in the visible region are of approximately the same intensity and shape but of opposite sign. A notable feature in the CD spectra of the two enzymes after light irradiation is the appearance of a CD band in the longer wavelength region (550-650 nm) and the opposite signs of the CD band in this region in the two enzymes. The similarity of the shape and intensity of the CD spectra of the two enzymes suggests that the environments surrounding the flavin moieties are very similar, and the sign reversal of the CD bands suggests that the mutual orientations between the transition moment of flavin and that of its environment differ in the two enzymes.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Cholesterol oxidase in microemulsion: Enzymatic activity on a substrate of low water solubility and inactivation by hydrogen peroxide

Cholesterol oxidase from Nocardia erythropolis, Pseudomonas, and Streptomyces species was active in microemulsion in which cholesterol is well solubilized. The activity was stable in nonionic microemulsions whereas in cationic and anionic microemulsions the activity decreased with time. The coupled activity test using horseradish peroxidase which is very stable in microemulsion, was modified. The activity at very low water concentration in nonionic microemulsions increased with the water content. The kinetic constants were determined: the Michaelis constant is in the range 10 to 28 mm in the microemulsions, compared to 10 to 28 μm in buffer. The maximum velocity was reduced by a factor of 3 to 5 compared to that in buffer. Neither substrate excess nor product inhibition was detected. The preparative oxidation of cholesterol revealed the inactivation of the cholesterol oxidase by hydrogen peroxide. In contrast to glucose oxidase, hydrogen peroxide inactivated cholesterol oxidase in the absence of substrate. Catalase provides protection during the cholesterol oxidation. Microemulsions are very good media in which to perform enzyme catalyzed reactions with substrates of low water solubility. Their use for the reproducible determination of cholesterol should be examined.     

Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune

To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity

The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.     

Sodium ascorbate improves yield of urinary steroids during hydrolysis with Helix pomatia juice

Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3beta-hydroxy-5-ene structure undergo transformation and yield of 5alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5alpha-reduced steroids is low due to oxidative damage of beta-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested.     

pKa values of the 8 alpha-imidazole substituents in selected flavoenzymes containing 8 alpha-histidylflavins

Difference absorption spectroscopy as a function of pH is described as a probe to determine the pKa values of the 8 alpha-imidazole substituent in flavoenzymes containing 8 alpha-histidylflavin coenzymes. Reversible absorption difference spectra are observed in the pH range 5.5 to 8.5 when synthetic 8 alpha-imidazolyl-FMN is bound to the apoflavodoxins from Azotobacter vinelandii and from Clostridium pasterianum. The observed spectral perturbations of these two flavodoxin complexes follow a single proton ionization dependence with respective pKa values of 6.7 and 6.8. No pH-induced spectral perturbations were observed when 8 alpha-(N-CH3)-imidazolium FMN was bound to either flavodoxin. Similar approaches are described to determine the 8 alpha-imidazolyl pKa values of the 8 alpha-histidyl-FAD coenzyme of the cholesterol oxidases from Schizophyllum commune and from Gleocystidium chrysocreas. Previous work has shown the former enzyme contains an 8 alpha-N1-histidyl-FAD (W. C. Kenney et al. (1979) J. Biol. Chem. 254, 4689-4690) while experiments reported here show the latter enzyme also contains one 8 alpha-N1-histidyl-FAD per mole of enzyme. The pKa value for the 8 alpha-imidazole substituent on the flavin of S. commune cholesterol oxidase is 5.4 while that determined for the G. chrysocreas enzyme is 6.2. These results demonstrate that the pKa of the 8 alpha-imidazole substituent can be determined in enzymes containing an 8 alpha-histidylflavin, provided that the enzyme is stable in the pH range required to observe ionization. Furthermore it is shown this the pKa value can differ even on comparison of enzymes from different sources that catalyze the same reaction.     

Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment

Hydrogen atoms are a vital component of enzyme structure and function. In recent years, atomic resolution crystallography (>or=1.2 A) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis. Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.     

Genetic designs for product formation in recombinant microbes

Several new bacterial host-vector systems for Klebsiella, Erwinia, Xanthomonas, Nocardia, and Streptomyces have been developed. With these host-vector systems, a strain of Klebsiella, which overproduces the extracellular starch-debranching enzyme, pullulanase, has been developed. The gene for cholesterol oxidase was cloned and used to develop a strain of Streptomyces lividans that extracellularly produces the enzyme, cholesterol oxidase, which is utilized to process cholesterol and diagnostically. The genes for these two enzymes were sequenced, and several interesting facts about their structures and secretory mechanisms were found. For expression of mammalian gene products, the expression vectors. pYM001 to pYM008, containing the lambda P(R)P(L) promoter, which is controlled by a thermolabile repressor, have been developed. The activities of these promoters were compared in various bacterial strains with the galK monitoring system. E. coli promoters, such as lac, trp, tac, lambda P(R), P(L), and P(R)P(L), were found to be expressed in other enteric bacteria and in Bacillus subtilis. With these expression vectors, the vesicular stomatitis virus-nucleocapsid, monkey metallothionein, and human apolipoprotein A1 genes were expressed in E. coli.     

Activity of microorganisms decomposing cholesterol]

The rates of cholesterol decomposition was compared among cell suspensions of different microorganisms. Fifty seven cultures were studied: 2 strains of actinomycetes, 23 strains of proactinomycetes, 22 strains of mycobacteria, and 10 strains of bacteria. During four hours of incubation, 11 strains virtually did not decompose cholesterol at all, 10 strains decomposed it by up to 20 per cent, 21 strains by 20 to 70 per cent, and 15 strains by 70 to 100 per cent. The highest activity was displayed by Mycobacterium rubrum 297 and Mycobacterium sp. 2B, which decomposed all cholesterol in the sample within 4 hours. The high activity was also displayed by Proactinomyces sp. 2006, Nocardia opaca JMET 7030, and Nacardia erythropolis JMET 7252.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

Identification of cholesterol oxidase from fast-growing mycobacterial strains and Rhodococcus sp.

Cholesterol oxidase synthesis ability by various fast-growing mycobacteria (M. fortuitum, M. vaccae, M. phlei, M. smegmatis) and Rhodococcus sp. IM 58 was investigated by thin-layer chromatography and Western blot analysis. In contrast with the case of cholesterol oxidases from Arthrobacter sp. IM 79 and Streptomyces sp., those from the strains tested were localized intracellularly. Western blot experiments revealed high antigenic similarity of those enzymatic proteins to cholesterol oxidase from Schizophyllum commune, and lack of homology to cholesterol oxidase from Streptomyces sp. Differences in the analyzed enzymes were due not only to a single antigenic determinant alteration but also to the significant distinctions on the genetic level as shown by Southern blot hybridization.     

A clone coding for Schizophyllum commune beta-glucosidase: homology with a yeast beta-glucosidase

Three identical clones coding for a partial sequence of the Schizophyllum commune beta-glucosidase were isolated from a cDNA library in lambda gt11, using polyclonal antibody to the enzyme. The identity was confirmed by comparison of the amino-terminus of a peptide from a protease lys-C digest with the sequence inferred from the cDNA sequence. A comparison of the sequence with that reported for a beta-glucosidase from Candida pelliculosa revealed a region in the latter with 43% identity in amino acid sequence. There was also a similarity in the S. commune beta-glucosidase to an active site sequence proposed for a S. commune endoglucanase, suggesting the possibility of a common catalytic mechanism for these two glucolytic enzymes.     

3-keto-5 alpha-steroid-delta 4-dehydrogenase from Nocardia corallina: purification and characterization

The inducible 3-keto-5 alpha-steroid-delta 4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5 alpha-steroid at the 4- and 5-position, e.g. the conversion of 5 alpha-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5 alpha-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4 beta, 5 alpha-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5 alpha-steroids but not 3 beta-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5 alpha-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-delta 1-dehydrogenase.     

Substrate specificity of the biotransformation enzymes in a Nocardia erythropolis culture

Substrate specificity of biotransformation enzymes of culture Nocardia erythropolis was studied. Products of transformation of cholesterol and three sterols of microbial origin: ergosterol, ergosta-5,7-dien-3 beta-ol and ergosta-7,22-dien-3 beta-ol was identified with a help of thin-layer chromatography, UV spectrophotometry and mass-spectrometry. It was established, that delta 22-bond in the side chains of sterols and delta 7-bond slows and delta 5-bond makes impossible cleavage of side chains of sterols.