Utilization of exocellular mannan from Rhodotorula glutinis as an immunoreactive antigen in diagnosis of leptospirosis.
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Previously, Rhodotorula glutinis was reported to produce a large amount of exocellular mannan, having a repeating unit of -->3)-D-Manp-(1-->4)-D-Manp-(1-->. Recently, we found that antigenic polysaccharides of Leptospira biflexa serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of Rhodotorula mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from R. glutinis AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and (1)H- and (13)C-labeled nuclear magnetic resonance spectrometry, the Rhodotorula mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the Rhodotorula mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis. (+info)
Redox potentials and their pH dependence of D-amino-acid oxidase of Rhodotorula gracilis and Trigonopsis variabilis.
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The redox potentials and pH characteristics of D-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5-8.5 at 15 degrees C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single-electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a approximately -60 mV/pH unit slope, consistent with a one-electron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (-30 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO-benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding. (+info)
The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.
(11/173)
Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases. (+info)
Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast.
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Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast. (+info)
Influence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca.
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The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast. (+info)
Molecular phylogenetics of the genus Rhodotorula and related basidiomycetous yeasts inferred from the mitochondrial cytochrome b gene.
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Phylogenetic relationships of basidiomycetous yeasts, especially of the genus Rhodotorula, were studied using partial sequences of the mitochondrial cytochrome b gene. The results demonstrated that the basidiomycetous yeasts under investigation distributed into two main clusters: one containing Tremellales, Filobasidiales and their anamorphs and the other containing Ustilaginales, Sporidiales and their anamorphs. This clustering in turn correlates with cell wall biochemistry, presence or absence of xylose, and septal ultrastructure, dolipore or simple pore. Bullera, Bulleromyces, Filobasidiella, Cryptococcus and Trichosporon, yeasts of the former cluster, contain xylose in the cell wall and have dolipore septa. In contrast yeasts of the latter cluster, which included Bensingtonia, Erythrobasidium, Leucosporidium, Malassezia, Rhodosporidium, Rhodotorula, Sporidiobolus, Sporobolomyces and Ustilago, have no xylose in the cell wall and have a simple pore septum. Yeasts of the latter group could be further divided into four clades (A-D). Species of Rhodotorula were distributed in all of these clades, indicating the polyphyletic nature of the genus. A limited number of Rhodotorula species demonstrated identical sequences, for example Rhodotorula bacarum and Rhodotorula foliorum, Rhodotorula fujisanensis and Rhodotorula futronensis, Rhodotorula glutinis var. dairenensis and Rhodotorula mucilaginosa. However, all the other test species of the genus Rhodotorula were well separated based on their 396 bp nucleotide sequences. These results demonstrate the effectiveness of the use of cytochrome b sequences for both species identification and the study of phylogenetic relationships among basidiomycetous yeasts. (+info)
Increased transglycosylation activity of Rhodotorula glutinis endo-beta-glucanase in media containing organic solvent.
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The transglycosylation of p-nitrophenyl-beta-D-cellotrioside to cellotetraose catalyzed by endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50 degrees C in the presence of 40% acetone. (+info)
pH and kinetic isotope effects in d-amino acid oxidase catalysis.
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The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis d-amino acid oxidase were investigated. The rate constant for enzyme-FAD reduction by d-alanine increases approximately fourfold with pH, reflecting apparent pKa values of approximately 6 and approximately 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both d-alanine and d-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with d-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate isotope effect on the reduction rate with [2-D]d-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with [2-D]d-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rupture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in d-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pKa,app of approximately 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha-amino group, but also includes contributions from kinetic parameters. (+info)