A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase.
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The apoenzyme of D-amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo-D-amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 x 10(-8) M was calculated. The kinetics of formation of the apoprotein-FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two-stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4-6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast D-amino acid oxidase appears to be of the type Apo + FAD in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out. (+info)
Epidemiology and outcome of Rhodotorula fungemia in a tertiary care hospital.
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We reviewed demographic data, risk factors, treatment, and outcomes associated with Rhodotorula fungemia in a tertiary care hospital during 2002-2005. Rhodotorula species caused fungemic episodes in 7 patients during the 4-year period that we studied. The most common predisposing factors were patients with hematological and solid malignancy receiving corticosteroids and cytotoxic drugs, the presence of central venous catheters, and the use of broad-spectrum antibiotics. Because of Rhodotorula species's intrinsic resistance to triazole and echinocandin antifungal agents, patients receiving fluconazole and caspofungin might be susceptible to the development of breakthrough Rhodotorula fungemia. (+info)
Mechanistic and active-site studies on D(--)-mandelate dehydrogenase from Rhodotorula graminis.
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D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--)-mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--)-bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(--)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway. (+info)
The distribution of pyrophosphatidic acid in nature.
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The occurrence of a novel phospholipid, pyrophosphatidic acid, in the lipid extracts of yeasts (23 species), bacteria (E. coli), algae (chlorella), mammalia (human, rabbit, guinea pig, and mouse), insect (cockroach), fish (carp), mollusc (clam), and spermatophyta (spinach) was investigated. Pyrophosphatidic acid was found exclusively in the lipid extracts of several kinds of yeast species, but not in other normal living species (animals, plants, and microorganisms) so far investigated. All of the yeast species containing this lipid belong to the asporogenous yeasts (Cryptococcus neoformans CBS-132, Cryptococcus laurentii Z 6-5, Rhodotorula glutinis H 3-9-1, Rhodotorula rubra AY-2, Kloeckera apiculata KK-3, and Trichosporon cutaneum KC 4-3), and ballistosporogenous yeast (Sporobolomyces salmonicolor WF 174). In contrast, no detectable amount of pyrophosphatidic acid was found in the cellular lipids of ascosporogenous yeasts. (+info)
Meningitis caused by Rhodotorula rubra in an human immunodeficiency virus infected patient.
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Rhodotorula spp. are common saprophytes but may be responsible for systemic infections in immunocompromised patients. Meningitis caused by Rhodotorula spp. in human immunodeficiency virus (HIV) infected patients has been reported only rarely. We present a case of meningitis caused by Rhodotorula rubra in HIV infected patient. The presumptive diagnosis of cryptococcal meningitis was made on the basis of India ink preparation, Gram staining and latex agglutination test (LAT) for cryptococcal antigen. The final diagnosis was confirmed by isolation of Rhodotorula rubra from cerebrospinal fluid on culture. LAT was considered false positive. Amphotericin B and 5-fluorocytosine were administered but the patient succumbed to his illness. (+info)
Rhodotorula subericola sp. nov., an anamorphic basidiomycetous yeast species isolated from bark of Quercus suber (cork oak).
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Two yeasts strains, Y-31(T) and Y-20B, pertaining to a previously unknown yeast species were isolated from bark of cork oak in Spain. Physiological characterization revealed a pattern of assimilation of carbon and nitrogen compounds compatible with members of the genus Rhodotorula. From sequence analysis of the D1/D2 region of the 26S rRNA gene, Rhodotorula cycloclastica and Rhodotorula philyla were related to the unknown species. Phylogenetic reconstruction based on the D1/D2 region of the 26S rRNA gene showed that the novel species clustered in a branch together with R. cycloclastica. The name Rhodotorula subericola sp. nov. is proposed, with isolate Y-31(T) (=CECT 11976(T)=CBS 10442(T)) the type strain of this novel taxon in the Microbotryum lineage, subclass Microbotryomycetidae, class Urediniomycetes of basidiomycetous yeasts. (+info)
Rhodotorula psychrophila sp. nov., Rhodotorula psychrophenolica sp. nov. and Rhodotorula glacialis sp. nov., novel psychrophilic basidiomycetous yeast species isolated from alpine environments.
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Three novel psychrophilic species of the genus Rhodotorula are described. Rhodotorula psychrophila sp. nov. (type strain PB19(T)=CBS 10440(T)=DSM 18768(T)), Rhodotorula psychrophenolica sp. nov. (type strain AG21(T)=CBS 10438(T)=DSM 18767(T)) and Rhodotorula glacialis sp. nov. (type strain A19(T)=CBS 10436(T)=DSM 18766(T)) were isolated from soil collected from an alpine railway area, from mud in the thawing zone of a glacier foot and from glacier cryoconite, respectively. All three species have been assigned to the genus Rhodotorula on the basis of molecular sequence data and physiological and morphological properties. Rhodotorula psychrophila is not able to grow at temperatures above 15 degrees C. Rhodotorula psychrophenolica and Rhodotorula glacialis degrade high concentrations of phenol (up to 12.5 and 5 mM, respectively) as the sole carbon source at 10 degrees C. Sequence analyses of the 26S rDNA D1/D2 regions indicated that the novel species are phylogenetically related and belong to a clade that includes other psychrophilic yeasts. (+info)
All eight possible mono-beta-D-glucosides of validoxylamine A. I. Preparation and structure determination.
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Validamycin A is the major and most active compound among the validamycin complex. Since the site of beta-glucosidic attachment to validoxylamine A (1) was expected to affect the activity against the pathogenic fungus, Rhizoctonia solani, all eight possible mono-beta-D-glucosides of 1 were prepared. 2-O-, 4-O-, 4'-O-, and 7'-O-beta-D-glucopyranosylvalidoxylamine A (2, 4, 6 and 9, respectively) were prepared by microbial beta-glycosylation of 1 with strains of Rhodotorula sp. 7-O- and 6'-O-beta-D-glucopyranosylvalidoxylamine A (5a and 8a, respectively) were prepared semisynthetically through microbial formation of 7-O-beta-D-glucopyranosylvalidamine (10), oxidation of the primary amine of 10 to a ketone, and coupling of the ketone derivative with valienamine, and through microbial formation of 6-O-beta-D-glucopyranosylvalienamine (11), and coupling of 11 with (2R)-(2,4/3,5)-2,3,4-trihydroxy-5-hydroxymethylcyclohexanone (12), respectively. 3-O- and 5'-O-beta-D-glucopyranosylvalidoxylamine A (3a and 7a, respectively) were chemically synthesized. (+info)