Intra- and extracellular forms of ethanol-modified O-underglycosylated galactose oxidase

The effect of ethanol and tunicamycin on synthesis and secretion of galactose oxidase was studied in resting cells of Dactylium dendroides. Ethanol promoted an overall decrease in both intra- and extracellular enzyme levels to the same extent that it inhibited [14C]glucosamine incorporation into total protein. The carbohydrate content of the intracellular enzyme was also depressed (44%) with a simultaneous decrease in O-Ser linked oligosaccharides. The intracellular galactose oxidase obtained after exposure of mycelia to ethanol plus tunicamycin lost 86% of its carbohydrate moieties, whereas the extracellular form lost only 35%. In both cases, residual sugar moieties were not eliminated by mild alkaline treatment. These data suggest that ethanol affects O-glycosylation of galactose oxidase. O-Underglycosylation did not affect the S0.5 values for galactose but diminished the molar catalytic activity. The absence of O-Ser/Thr-linked saccharides turned the intracellular enzyme into a form more susceptible to proteolysis than that devoid of N-linked sugars (tunicamycin-treated). O-Underglycosylation had a significant effect on the renaturation-reactivation of the enzyme after denaturation with 2.4 M Gdn-HCl.     

Role of carbohydrate content on the properties of galactose oxidase from Dactylium dendroides

The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.     

Regulation of galactose oxidase synthesis and secretion in Dactylium dendroides: effects of pH and culture density.

The effects of pH and growth density on the amount of an extracellular enzyme, galactose oxidase, synthesized by the fungus Dactylium dendroides were studied. Growth at a pH below 6.7 caused a decrease in the ability of the organism to release galactose oxidase. The enzyme retained by these fungal cells was liberated whenever the pH was raised to 7.0. Cycloheximide addition failed to inhibit the appearance of this protein; [3H]leucine added prior to pH adjustment was not incorporated into the released protein, These observations indicate the released protein is not newly synthesized protein. The retained enzyme would be secreted slowly over a 2-day period if the pH was not increased. In addition to regulating protein retention, pH was also shown to be associated with vacuolization, cell volume, culture density, and inhibition of protein synthesis. Cultures maintained at low pH were characterized by a dense growth consisting of highly vacuolated, buoyant, fungal hyphae. Increasing the pH from 6 to 7 caused a decrease in vacuole size. Cells grown at neutral pH maintained a lower density of growth and, based on activity measurements, synthesized 33% more galactose oxidase. Furthermore, cultures grown at pH 6.0 and maintained at a lower cell density produced galactose oxidase at a level similar to that of cells grown at neutral pH. Thus, the elevated density of the cell culture was inhibitory to galactose oxidase synthesis. The observed effects on protein synthesis and release were rather specific for galactose oxidase, since other extracellular proteins appeared in the earliest stages of growth.     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Re-examination of the products of the action of galactose oxidase. Evidence for the conversion of raffinose to 6'-carboxyraffinose

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose to an aldehyde group. When the products of a galactose oxidase-catalase treatment of raffinose were examined by gel filtration and ion exchange chromatography, we found that, in addition to the expected 6'-aldehydoraffinose, two other components were present. Of these two components, the major one was retained on a column of AG 1-X8 (formate), gave a positive carbazole reaction for uronic acid, and on paper chromatograms had a mobility identical with that of 6'-carboxyraffinose. The infrared spectrum of the compound showed a carbonyl absorbance at 1725 cm-1 and was distinguishable from the spectra of raffinose and 6'-aldehydoraffinose. These data showed that raffinose was partly converted to 6'-carboxyraffinose when treated with galactose oxidase and catalase. The conversion of [3H]raffinose to [3H]6'-carboxyraffinose increased gradually with time of oxidation from 22% at 6 h to 68% at 96 h. Results of other experiments provided evidence that this was an enzymic conversion and depended on the presence of galactose oxidase. The activities responsible for the formation of aldehyde and uronic acid could not be separated by affinity chromatography, gel electrophoresis, or ion exchange chromatography, indicating that the same enzyme is responsible for both activities. Treatment of galactose, melibiose, and stachyose with galactose oxidase and catalase also resulted in the formation of the corresponding uronic acids. These studies indicate that galactose oxidase not only converts the C-6 hydroxymethyl group of galactose to an aldehyde group, but also catalyzes further oxidation to the carboxyl group.     

Stereospecific oxidation of aliphatic alcohols catalyzed by galactose oxidase

The stereospecificity of galactose oxidase (EC 1.1.3.9) from Dactylium dendroides in the oxidation of simple three-carbon alcohols has been examined. The enzyme oxidizes glycerol to optically pure S(?)glyceraldehyde. In addition to this prochiral stereospecificity, galactose oxidase also exhibits enantiomeric stereospecificity in the oxidation of 3-halo-1,2-propanediols: the R isomer appears to be a better substrate than its S counterpart. The above stereochemistry of galactose oxidase-catalyzed oxidation of “unnatural” substrates, non-sugar alcohols, can be predicted on the basis of the conformation of the natural substrate of the enzyme, D-galactose.     

Various properties of galactose oxidase from Fusarium graminearum IMV-F-1060 immobilized on aminoorganosilochromes

Galactose oxidase preparations are obtained from Fusarium graminearum IMV-F-N 1060 immobilized on aminoorganosilochromes activated by cyanuron chloride and 2.4-toluylene diizocyanate. The immobilized preparations were studied for their selective action on different carbohydrate substrates and for the pH-medium dependence of the obtained preparation activity. Potassium ferricyanide is established to have an activating effect on the immobilized enzyme. It is shown that the immobilized galactose oxidase preparations may be used for the analysis of galactose and lactose.     

Production of Galactose Oxidase by Gibberella fujikuroi

The distribution of galactose oxidase was investigated among microorganisms. Gibberella fujikuroi excreted a large amount of the enzyme into the culture medium. A study of the cultural conditions of this organism for enzyme formation showed that galactose oxidase production required copper. Copper was found to play a role, not only in the conversion of apoenzyme to holoenzyme, but also in regulation of the biosynthesis of galactose oxidase protein.     

Isolation and screening of glucose oxidase producing microorganisms from natural sources.

Among 1486 mould strains isolated from natural sources (screened for extracellular glucose oxidase) only 119 (Aspergillus and Penicillium) showed this enzyme activity. As the best glucose oxidase producer, A. niger 0-1 was isolated from decaying tree. The dynamics of glucose oxidase synthesis in A. niger 0-1 during its culture by submerged method show that the intracellular activity of this enzyme is 10-times higher than its extracellular level. Some properties of the crude glucose oxidase preparation, isolated from the postculture liquids by lyophilization, were examined.     

Stereoselective hydrogen abstraction by galactose oxidase

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-alpha-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 alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.     

A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

Sugar compositions of extra- and intracellular polysaccharides produced by candida lipolytica (4124) grown on n-hexadecane

The free monosaccharide content of C. lipolytica (strain 4 124) cells grown on n-hexadecane was identified and found to be only glucose. The chromatographic analysis of the hydrolysate of intracellular cell wall polysaccharides indicated the presence of glucose: mannose: galactose: xylose in a ratio of 1 : 1.32 : 1.07 : 0.35. Paper and dise electrophoresis of extracellular polysaccharid from the culture broth was found to be heterogeneous. Ethanol fractionation separated it to a major component F (I) 81.99% and a minor one F (II) 13.04%. Analysis of the major fraction showed that it consisted of galactose and mannose only while the minor polysaccharide consisted of galactose, glucose and mannose. Thus it was concluded that the predominant sugar in both extracellular and intracellular polysaccharides was mannose. Xylose was detected in the intracellular polysaccharide only.     

Intracellular and extracellular beta-N-acetylhexosaminidases from Physarum polycephalum. Comparison of vegetative and spherulation enzymes.

Vegetative microplasmodia of the slime mold, Physarum polycephalum, produce an intracellular β-N-acetylhexosaminidase enzyme when grown on a medium containing 1% glucose, 0.15% yeast extract, and 1% peptone. When early log-phase microplasmodia are induced to differentiate to spherules by starvation in a salts medium, they excrete an extracellular β-N-acetylhexosaminidase. Both of these enzymes have been purified to apparent homogeneity. Characterization studies showed that the extracellular enzyme was nonidentical to the preexisting, vegetative enzyme and the enzyme in completed spherules. Evidence demonstrating dissimilarities between the two proteins included marked differences in (i) specificities for several natural and synthetic substrates, (ii) various kinetic parameters, (iii) relative net charges as evidenced by different elution behavior from similar DE-52 cellulose chromatography columns, (iv) carbohydrate contents, and (v) subunit polypeptide molecular weights. Conclusive evidence for their nonidentity was shown in their respective amino acid compositions and divergent immunological properties. The extracellular β-N-acetylhexosaminidase demonstrated a subunit molecular weight of 25,300; the intracellular enzyme subunit molecular weight was 40,500. The extracellular enzyme, with the smaller polypeptide subunit, contained 1.79 times as many aromatic amino acid residues in tyrosine, phenylalanine, and tryptophan as the intracellular enzyme. Thus, the extracellular enzyme could not have been comprised of subunits derived from limited proteolytic hydrolysis of the larger subunits of the intracellular enzyme. Rabbit antisera prepared against each purified β-N-acetylhexosaminidase failed to yield precipitin bands with the heterologous antigen in immunodiffusion tests. Thus, apparently distinct structural genes code for these two enzymes and they may serve different, but unidentified, physiological functions.     

Purification of galactose oxidase from Dactylium dendroides by affinity chromatography on melibiose-polyacrylamide

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose and galactosamine to an aldehyde group. It has been widely used in glycoconjugate research, for example in the labeling of asialoglycoproteins. We have developed a simple affinity purification for galactose oxidase using melibiose-polyacrylamide. This affinity procedure was used to purify the enzyme from ammonium sulfate precipitates of culture filtrates of Dactylium dendroides. The material containing proteases and other contaminants is eluted in the buffer wash. The galactose oxidase is then specifically eluted from the column with buffer containing 0.1 M D-fucose or D-galactose. Using this procedure, the enzyme was also purified from commercial samples of galactose oxidase which contain high proteolytic activity.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Activation of galactose-containing glycoprotein and solid supports by galactose oxidase in presence of catalase for immobilization purposes

Summary Specific oxidation of D-galactose present in the carbohydrate moiety of glucose oxidase from Aspergillus niger by galactose oxidase in the presence of catalase (48% efficiency) did not change the activity of the enzyme. Oxidized enzyme was coupled to hydrazide derivatives of O--D-galactosyl Separon H 1000 or of Sepharose 4B. Both solid supports were modified with adipic acid dihydrazide after their activation with galactose oxidase. Each immobilized preparation of glucose oxidase showed higher activity than was achieved by other immobilizing procedures.     

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.     

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.     

Properties of Fusarium graminearum galactose oxidase

The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.     

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.