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G M Shavlovskiĭ

Publications and source records attributed to G M Shavlovskiĭ.

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

[Biosynthesis of 6,7-dimethyl-8-ribityllumazine in yeast extracts of Pichia guilliermondii].

The effects of some hexoses, pentoses, their phosphoric esters, ribitol and pyruvate on the synthesis of 6.7-dimethyl-8-ribityllumazine in extracts of prototrophic strain and riboflavin-deficient mutants of the yeast Pichia guilliermondii ATCC 9058 were studied. Incubation of dialyzed cell extracts of the mutant strain RG123 (rib7, his-) with blocked riboflavin syntase in the presence of 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate, NADPH and ribose-5-phosphate results in production of 6.7-dimethyl-8-ribityllumazine. In similar experiments with cell extracts of the prototrophic strain 6.7-dimethyl-8-ribityllumazine and riboflavin were formed. Consequently all the reactions of vitamin B2 biosynthesis occurred under these conditions. Glucose 6-phosphate and fluctose 6-phosphate weakly activated the biosynthesis of 6.7-dimethyl-8-ribityllumazine, while ribose, ribitol, fructose, glucose and pyruvate had no effect on this process. When cell extracts of mutant strains of genotypes rib5 and rib6 with a blocked enzyme(s) of the fourth step of flavinogenesis were incubated in the presence of ribose 5-phosphate no 6.7-dimethyl-8-ribityllumazine formation took place. It is concluded than in the P. guilliermondii yeast ribose 5-phosphate or its metabolite can donate carbon atoms for 6.7-dimethyl-8-ribityllumazine biosynthesis from its pyrimidine precursor.

Ascomycota↗
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↗
PubMed1981

[Riboflavin transport in mutant of Pichia guilliermondii yeast with damaged glucose uptake].

The mutant of P. guilliermondii yeast MS-50 which had lost the ability to grow in a glucose-containing medium but not in the presence of sucrose or maltose was selected. The mutant has a damaged glucose uptake from the medium. The riboflavin permease activity of the MS1-50 mutant is not impaired; glucose inhibits the riboflavin uptake by this strain. Thus, riboflavin permease does not promote glucose transport into yeast cells.

Ascomycota↗
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

[Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast].

The interaction of purified riboflavin kinase (EC 2.7.1.26) from Pichia guilliermondii with 44 structural vitamin B2 analogues is studied. The presence of D-ribityl lateral chain in an analogue structure is found to be necessary for the substrate activity. The substitution of CH3 groups in the 7 and 8 positions of isoalloxazine ring in the riboflavin molecule for CF3, Cl, H, NH2 and N(CH3)2 resulted in the decrease of the analogue affinity to riboflavin kinase as compared with the natural substrate, vitamin B2. The most efficient enzyme inhibitors of analogues without substrate properties turned to be trifluoromethylisoalloxazines, containing 2'-hydroxyethyl group at N10. The elongation of D-ribityl lateral chain, the elimination of change of CH3-groups in the 7 and 8 positions for CF3- Cl-, COOH-substitutors resulted in the decrease of the inhibitory effect of flavines. Modifications in the structure of isoalloxazine ring, etherification of OH-groups in the lateral D-ribityl chain, and the introduction of volume substitutors (N-piperidyl, D-ribitylamine, hydroxyethylamine) prevented the interaction of the analogue with riboflavin kinase. Flavin nucleotides (FMN and FAD) did not affect the rate of vitamin B2 phosphorylation.

Ascomycota↗
PubMed1978

[Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii].

Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.

Ascomycota↗
PubMed1978

[Regulation of the activity of GTP-cyclohydrolase, the enzyme of the first step of flavinogenesis in yeasts].

The kinetic properties and regulation of activity of GTP-cyclohydrolase, the enzyme of the first step of flavinogenesis in the Pichia guilliermondii yests, partially purified by gel-filtration were studied. It was found that the curve of the dependence of reaction rate on substrate concentration is non-hyperbolic. FAD inhibited the enzyme activity, while riboflavin and FMN had no such effect. In addition to FAD, 5'-AMP, 3',5'-AMP, ADP, ATP, NAD and NADP inhibited the enzyme activity. Under combined action of FAD and AMP on GTP-cyclohydrolase no synergetic or antagonistic effects of the inhibitors on the enzyme activity were observed. The enzyme appreciably lost its sensitivity to FAD and AMP after thermal treatment. The data obtained suggest that GTP-cyclohydrolase from P. guilliermondii is an allosteric enzyme, which is inhibited by the end product of flavinogenesis FAD, as well as by other 5'-AMP-containing nucleotides.

Adenine Nucleotides↗
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↗
PubMed1977

[Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants].

Riboflavin deficient mutant Pichia guilliermondii MS1 which requires approximately 1000-fold lower concentration of exogenous vitamin B2 for growth when compared with a non-adapted riboflavin deficient mutants of this species was isolated by means of of UV-irradiation. The growth of the mutant was strongly inhibited by actinomycin D and L-canavanine. The revertant MS8 and MS14 which synthesized riboflavin were selected from the strain MS1. These revertants posses a multiple sensitivity to actinomycin D, rifamycin, euflavine, mitomycin C, antimycin A, 8-azaadenine, 8-azaguanine, L-canavanine and 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine. The ability to utilized glycerol and ethanol as a sole carbon source for growth was impaired in these mutants. The mutants which can utilize glycerol were isolated from the strain MS14. Such mutants were resistant to actonomycin D. Mutation (s) which determines a multiple sensitivity and inability to utilized glycerol was recessive.

Anti-Bacterial Agents↗
PubMed1977

[Active transport of riboflavin in the yeast Pichia guilliermondii. Detection and some properties of the cryptic riboflavin permease].

In contrast to cells and protoplasts of the wild strain intact cells and protoplasts of riboflavin (RF)-deficient mutants of Pichia guilliermondii yeast possessing multiple sensitivity to antibiotics and antimetabolites were found capable to accomplish active transport of RF. The accumulation of RF against concentration gradient was energized by endogenous energy sources and was strongly depressed by uncouplers of oxidative phosphorylation and by inhibitors of respiration. RF transport was also blocked by the agents which destroy the permeability barrier and by sucrose. Cyclohemide did not inhibit the transport of vitamin B2. Uptake of RF depends on temperature and pH of the incubation mixture. RF permease possessed rather stringent substrate specificity and did not catalyze transport of FMN and FAD. RF uptake in two strains tested, MS1-3 and MS1, followed saturation kinetics (Km = 0.17 mM and 2.0 mM, respectively) and was not connected with RF auxotrophy. The ability for exogenous RF uptake was controlled by recessive allele. Restoration of normal resistance to antibiotics and antimetabolites was accompanied by a decrease in the permease affinity for substrate.

Alleles↗