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E M Logvinenko

Publications and source records attributed to E M Logvinenko.

At least 19 recordsLinked to original sources
PubMed1993

[Cloning of the RIB7 gene encoding the riboflavin synthase of the yeast Pichia guilliermondii].

The RIB7 gene encoding the enzyme of the final stage of riboflavin biosynthesis in Pichia guilliermondii--riboflavin synthase was cloned on the pFL38 shuttle vector as the Sau3A fragment of the chromosomal DNA of about 4 kb. The HindIII fragment of 1.4 kb was subcloned from the hybrid plasmid pFR7 obtained onto the pUC18 plasmid. The plasmid pR7 thus constructed transform Escherichia coli ribB-45 mutant cells with a blocked riboflavin synthase approximately at the same frequency as pFR7. High riboflavin synthase activity was discovered in the E. coli transformants carrying pR7 but not pFR7. Using both plasmids we also complemented rib17 mutant of P. guilliermondii.

Cloning, Molecular↗
PubMed1990

[Cloning of the RIB1 gene coding for the enzyme of the first stage of flavinogenesis in the yeast Pichia guilliermondi, GTP cyclohydrolase, in Escherichia coli cells].

The RIB1 gene encoding the enzyme of the first stage of the yeast Pichia guillermondii-GTP-cyclohydrolase- was cloned on pFL38 shuttle vector as the Sau3A fragment of chromosomal DNA of about 9 kb. EcoRI fragment of 4 kb with RIB1 gene was subcloned from the pFRI hybrid plasmid obtained into the pUC18 plasmid and then shortened to give 2.9 kb via deletion in SalGI site. The plasmid constructed was designated pR1. Activity of GTP-cyclohydrolase was 80-100-fold higher in extracts of transformants than in the prototroph strain, which evidence of effective expression of the yeast gene within recombinant plasmids in the cells of this species of bacteria. The enzyme isolated from transformants has molecular mass 179 kDa, is inhibited by PAD and adenyl-nucleotides, which is characteristic of GTP-cyclohydrolase of P. guilliermondii but not of Escherichia coli.

Adenosine Monophosphate↗
PubMed1987

[Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii].

The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively. It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein). No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant. Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene).

Genes, Fungal↗
PubMed1985

[Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81].

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.

Ascomycota↗
PubMed1983

[Purification and properties of GTP-cyclohydrolase of the yeast Pichia guilliermondii].

GTP-cyclohydrolase was isolated from the Fe-deficient cells of Pichia guilliermondii and purified 440-fold by treatment of extracts with streptomycin sulfate as well as by protein fractionation with (NH4)2SO4 at 25-45% saturation, gel filtration through Sephadex G-200 and DEAE-cellulose chromatography. The curves for the dependence of specific activity of GTP-cyclohydrolase on substrate and cofactor concentrations are non-hyperbolic; the values of [S]0.5 for GTP and Mg2+ are 2.2 X 10(-5) and 2 X 10(-4) M, respectively. The enzyme activity is inhibited by pyrophosphate ([I]0.5 = 5.8 X 10(-4) M), orthophosphate ([I]0.5 = 4.5 X 10(-3) M), heavy metal ions and chelating agents. The temperature optimum for the enzyme activity lies at 42-45 degrees C. The enzyme is labile at 4 degrees C but can well be stored at -15 degrees C. The pyrimidine product of the cyclohydrolase reaction, 2.5-diamino-6-oxy-4-ribosyl-aminopyrimidine-5'-phosphate, as well as pyrophosphate were purified from the reaction medium and identified.

Aminohydrolases↗
PubMed1982

[Effect of iron, actinomycin D and cycloheximide on the GTP-cyclohydrolase synthesis in flavinogenic yeasts].

The effect of Fe on the GTP-cyclohydrolase activity of the yeasts Pichia guilliermondii ATCC 9058 and Torulopsis candida BKM 13 whose flavinogenesis is controlled by Fe was investigated. The GTP-cyclohydrolase activity of yeast cells grown in an iron-deficient medium was 40-50 times that of the cells grown in an iron-rich medium. In the latter case the incubation of cells with alpha, alpha'-dipyridyl or 8-oxyquinoline also increased the enzyme activity. Cycloheximide prevented the rise in the cyclohydrolase activity in both cases, thus suggesting the participation of Fe in the control of the enzyme synthesis. Actinomycin D inhibited the enzyme derepression induced by alpha, alpha1-dipyridyl or 8-oxyquinoline in the P. guilliermondii MS1-37 mutant possessing a high sensitivity to this antibiotic. It is assumed that Fe is involved in the control of GTP-cyclohydrolase synthesis in flavinogenic yeasts at the transcription level.

Aminohydrolases↗
PubMed1980

[Detection of phosphorylated pyrimidine precursors of riboflavin in yeasts].

The nature of products formed from GTP in the presence of NADPH under effects of dialyzed extracts of the prototrophic strain of Pichia guilliermondii ATCC 9058 and mutants rib2 and rib3 with blocked second and third steps of flavinogenesis, respectively, was studied. The unstable pyrimidine intermediates were identified on the basis of the nature of pteridines formed upon condensation of riboflavin precursors with diacetyl. It was shown that under the action of extracts of the mutants rib3 GTP was predominantly converted into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate. In similar studies on the extracts of the prototrophic strain, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine phosphate, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine and trace amounts of 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine were detected. No ribitylated intermediates of riboflavin were formed in the extracts of rib2 mutants. The results obtained are indicative of the participation of phosphorylated pyrimidine precursors in the second and third steps of flavinogenesis in yeasts. 6,7-Dimethyl-8-ribityllumazine phosphate is not the substrate for P. guilliermondii riboflavin synthase.

Ascomycota↗
PubMed1979

[Genetic classification of riboflavin-dependent mutants of Pichia guilliermondii yeasts].

114 riboflavinless mutants were selected from the genetic line of Pichia guilliermondii yeast. By means of accumulation test the mutants were divided into five biochemical groups. In genetic experiments seven complementation classes were found among 106 mutants. The strains of the I biochemical group, accumulating no specific products, corresponded to complementation class rib1; II group, accumulating 2,4,5-triaminopyrimidine - to the class rib2; III group, accumulating 2,6-dihydroxy-4-ribitylaminopyrimidine - to the class rib3; the mutants of the IV group, accumulating 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine, were divided into three complementation classes rib4, rib5 and rib6; the mutants of the V group, acculumating 6,7-dimethyl-8-ribityllumazine, corresponded to the class rib7. Two mutants of the IV biochemical group within complementation classes rib4 and rib5 were detected could not grow in the medium with diacetyl without riboflavin. Intragenic complementation was found within classes rib6 and rib7. No linkage between mutations of different complementation classes was detected.

Ascomycota↗
PubMed1978

Origin of the ribityl side-chain of riboflavin from the ribose moiety of guanosine triphosphate in Pichia guilliermondii yeast.

In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.

Diacetyl↗
PubMed1977

[Activities of some yeast flavogenic enzymes in situ].

Effects of digitonin, dimethylsulfoxide and protamine sulfate on yeast Pichia guilliermondii were studied in order to produce cells with increased permeability and possessing the GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase activities. The digitonin-treated cells exhibited a higher cyclohydrolase activity than the cell-free extracts; the activities of riboflavinsynthetase and riboflavinkinase in the cells and cell-free extracts were found to be similar. Treatment of cells with dimethylsulfoxide proved to be most effective to determine the activity of GTP-cyclohydrolase and also helpful to determine that of riboflavinsynthetase. Protamine sulfate had no effect on the cells of P. guilliermondii. The methods developed were used to determine the activities of GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase in the cells of flavinogenic (P. guiller-mondii, Torulopsis candida) and non-flavinogenic (Candida utilis, Candida pulcherrima) yeasts grown in iron-rich and iron-deficient media. Derepression of riboflavinsynthetase and GTP-cyclohydrolase syntheses under conditions of Fe deficiency in the flavinogenic yeast cells confirmed previously made assumptions.

Aminohydrolases↗
PubMed1976

Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast.

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.

2,2'-Dipyridyl↗
PubMedDate not supplied

[Mechanism of retroinhibition in e regulation of flavinogenesis in yeasts of genus Pichia].

The kinetics of the synthesis of a riboflavin (RF) precursor, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine (DHARAP), was studied using the washed cells of RF-deficient mutants of Pichia guilliermondii R7G and Pichia ohmeri R32 with blocked lumasine synthetase. RF inhibited the synthesis of DHARAP while cycloheximide in the absence of RF had no effect on this process. The data suggest that flavins regulate the biosynthesis of RF in P. guillier mondii and P. ohmeri b7 means of feed-back inhibition mechanism.

Ascomycota↗
PubMedDate not supplied

[Nature of riboflavin precursors in Pichia guilliermondi yeasts].

Thirty-nine riboflavin-deficient mutants have been isolated from three yeast strains of Pichia guilliermondii (ATSS 9058, VKM Y-1256, VKM Y-1257) and F5-121 mutant which is capable of production of large amounts of riboflavin in the presence of iron in the medium. All mutants were divided into five groups according to the nature of precursors accumulated in the medium and growth reaction in media with 6,7-dimethyl-8-ribityllumasine and diacetyl. The mutants of the first group did not accumulate specific precursors of riboflavin either in the cells or in the medium. The mutants of the second, third and fourth groups accumulated, after the incubation with diacetyl, 2-amino-4-hydroxy-6,7-dimethylpteridine, 2-amino-4-hydroxy-6,7-dimethyl-8-ribitylpteridine and 6,7-dimethyl-8-ribityllumasine; therefore, they synthesized the following precursors of riboflavin: 2,4,5-triamino-6-hydroxy-pyrimidine, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine. The mutants of the fifth group accumulated 6,7-dimethyl-8-ribityllumasine in the medium and lacked riboflavin synthetase activity, as was confirmed by enzymatic studies.

Ascomycota↗