[Flavinogenesis and regulation of purine biosynthesis de novo in Pichia guilliermondi mutants resiatant to 8-azaguanine]. / Osobennosti flavinogeneza i reguliatsii biosinteza purinov de novo u mutantov drozhzhei Pichia guilliermondii, rezistentnykh k 8-azaguaninu

93 mutants resistant to 8-azaguanine (AGR-mutants) were derived from the strain of Pichia guilliermondii with blocked guanine deaminase (EC 3.5.4.3.) by UV-irradiation. The mutants retained the ability to uptake 8-azaguanine and guanine but could not deaminate guanine. Some of the AGR-mutants were found to accumulate large amounts of hypoxanthine and small amounts of guanine in the cultural medium. The inhibitory effect of guanine and 8-azaguanine but not adenine on the purine biosynthesis de novo was considerably decreased. It was established observing the rates of 5 amino 4-imidazoleribotide accumulation in purine-requiring AGR-mutants in the presence of different purines. The regulation of the activity and biosynthesis of IMP-dehydrogenase (EC 1. 2. 1. 14) with guanine compounds in AGR-mutants was completely preserved. Under cultivating in iron-rich medium all the AGR-mutants accumulated more riboflavin than the strain H-101 and the wild type strain. That occured as a result of the increase of flavinogenesis velocity in AGR-mutants during late logarithmic and negative growth acceleration phases. Some of mutants also synthesized more riboflavin in iron-deficient medium. Depression of riboflavine synthetase was not observed in the iron-rich cells of AGR-mutants.

[Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants]. / Mutanty drozhzhei Pichia guilliermondii s mnozhestvennoi chuvstvitel'nost'iu k antibiotikam i antimetabolitam. Soobshchenie I. Selektsiia i nekotorve svoistva mutantov.

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.

[Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii]. / O biokhimicheskikh funktsiiakh produktov genov RIB5 i RIB6, uchastvuiushchikh v biosinteze riboflavina u drozhzhei 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).

[Genetic classification of riboflavin-dependent mutants of Pichia guilliermondii yeasts]. / Geneticheskaia klassifikatsiia riboflavinzavisimykh mutantov drozhzhei Pichia guilliermondii.

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.

[Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81]. / Geneticheskii kontrol' biosinteza riboflavina u drozhzhei Pichia guilliermondii. Obnaruzhenie novogo reguliatornogo gena RIN81.

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.

[Cloning of the RIB7 gene encoding the riboflavin synthase of the yeast Pichia guilliermondii]. / Klonirovanie gena RIB7, kodiruiushchego riboflavinsintazu drozhzhei 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 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]. / Klonirovanie gena RIB1, kodiruiushchego ferment pervogo étapa flavinogeneza u drozhzhei Pichia guilliermondi--GTF-tsiklogidrolazu, v kletkakh Escherichia coli.

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.

[Supersynthesis of flavins in microorganisms and its molecular mechanism (review of the literature)]. / Sverkhsintez flavinov u mikroorganizmov i ego molekuliarnye mekhanizmy (obzor).

The present review discusses the significance of fundamental research into regulation of flavin biosynthesis for development of the knowledge about mechanisms of overproduction of these compounds and their manufacturing. The pathways of riboflavin, FMN and FAD biosyntheses and their regulation in some bacteria, yeasts and fungi are considered, as well as the recent advances in flavin biotechnology. The modern trends in microbial and enzymatic production of flavins are discussed.

[Flavin biogenesis in yeasts]. / Biogenez flavinov u drozhzhei.

The biosynthesis of flavins and methods for its study in yeast are considered. The chemical structure of flavin precursors and enzymes catalyzing certain stages of GTP transformation into riboflavin and flavin nucleotides are characterized. Differences in formation of flavins in bacteria and yeast are shown as well as possible ways of further research in this field.

[Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo]. / Ob ingibirovanii shchelochoni fosfatazy I drozhzhei Pichia guilliermondii in vitro i in vivo.

The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed.

[GTP-cyclohydrolases of microorganisms and their role in metabolism]. / GTP-tsiklogidrolazy mikroorganizmov i ikh rol' v obmene veshchestv.

The properties, regularities of biosynthesis and biochemical functions are considered of GTP-cyclohydrolases of microorganisms. The existence of two groups of these enzymes is established. The first group enzymes convert GTP into 7,8-dihydroneopterin-triphosphate and formiate. They participate in biosynthesis of tetrahydrofolic acid, tetrahydrobiopterin, molybdenic cofactor, pyrrolopyrimidine antibiotics and in a series of pigments. Representatives of the second group of cyclohydrolases convert GTP into 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate, formiate and pyrophosphate. They catalyze the first stages of formation of 6,7-dimethyl-8-ribityllumazine, flavins and their derivatives, toxoflavin (azapteridine antibiotics). The regulation of biosynthesis and activity of GTP-cyclohydrolases is studied only for individual enzymes of this group.

[Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide]. / Ochistka i svoistva shchelochnoi nukleotidpirofosfatazy drozhzhei Pichia guilliermondii, gidrolizuiushchei flavinadenindinukleotid.

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).

[Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide]. / Ochistka i svoistva kisloi nukleotidpirofosfatazy drozhzhei Pichia guilliermondii, gidrolizuiushchei flavinadenindinukleotid.

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.

[The nature of the pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase participating in riboflavin biosynthesis in yeasts]. / O prirode pirimidinovogo substrata 6,7-dimetil-8-ribitilliumazinsintazy, uchastvuiushchei v biosinteze riboflavina u drozhzhei.

2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine-5'-phosphate are studied for their effect on the activity of 6,7-dimethyl-8-ribityllumazine synthase of Pichia guilliermondii yeasts. It is shown that when nonphosphorylated form of pyrimidine and ribose-5-phosphate (donor C-4--a fragment) is used as a substrate, the specific activity of 6,7-dimethyl-8-ribityllumazine synthase is high and Be2+ and F- ions, inhibitors of alkaline phosphatases, do not inhibit it. The value of Km for this pyrimidine is 1.1 X 10(-5) M. Phosphorylated pyrimidine being used as a substrate in the presence of Be2+ and F-, the reaction practically does not proceed. Therefore, only 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine is a pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase of yeast.

[Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts]. / Svoistva fermenta vtorogo étapa flavinogeneza 2,5-diamino-4-oksi- 6-ribozilaminopirimidin-5'-fosfatreduktazy drozhzhei Pichia guilliermondii.

2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B. The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification. It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M. The reaction proceeds in the presence of NADPH or NADH. The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M). The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD. EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity. The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C. The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces klöckeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis. Aspergillus nidulans, Neurospora crassa (nonflavinogenic species). The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions.

[Regulation of the activity and synthesis of enzymes participating in the formation of 6,7-dimethyl-8-ribityllumazine, a riboflavin precursor in yeast]. / Reguliatsiia aktivnosti i sinteza fermentov, uchastvuiushchikh v obrazovanii predshestvennika riboflavina-- 6,7-dimetil-8-ribitilliumazina u drozhzhei.

The properties of two flavinogenesis enzymes--synthase of the aliphatic precursor of riboflavin (APR-synthase) and 6.7-dimethyl-8-ribityllumazinesynthase (DMRL-synthase) of Pichia guilliermondii. It is established that DMRL-synthase, uses APR as a substrate which contains, evidently, a phosphate group. The value of Km for APR is equal to 0.7.10(-5) M, for 2.4-dihydroxy-5-amino-6-ribitylaminopyrimidine--1.25.10(-5) M. It is riboflavin but not FAD that inhibits the activity of DMRL-synthase; the value (I)0.5 is equal to 2.10(-5) M. DMRL, riboflavin, flavin mononucleotide and FAD do not affect the APR-synthase activity. In iron-deficient cells of P. guilliermondii, Torulopsis candida, Debaryomyces klöckeri and Schwanniomyces occidentalis realizing the oversynthesis of riboflavin there occurs derepression of DMRL-synthase and APR-synthase.

[Localization of the genes coding for GTP cyclohydrolase II and riboflavin synthase on the chromosome of Escherichia coli K-12]. / Lokalizatsiia genov, kodiruiushchikh GTF-tsiklogidrolazu II i riboflavinsintazu na khromosome Escherichia coli K-12.

A conjugation analysis of riboflavin-dependent mutants of Escherichia coli K-12 has been made by means of various F'-factors. It is shown that the GTP cyclohydrolase II gene is localized in the chromosome map site between 27 and 30 min; the riboflavin synthase gene--in the site between 56 and 59 min; and the gene in which mutation causes accumulation of 2,6-dioxy-5-amino-4-ribitylamino-pyrimidine--in the site limited by 61-65 min. So it may be concluded that riboflavin biosynthesis genes in the E. coli chromosome are not clustered.

[The activity of enzymes involved in synthesis and hydrolysis of flavin adenine dinucleotide is Pichia guilliermondii studies at different levels of flavinogenesis]. / Issledovanie aktivnosti fermentov, uchastvuiushchikh v sinteze i gidrolize flavinadenin-dinkleotida u drozhzhei Pichia guilliermondii pri razlichnykh urovniakh flavinogeneza.

The activity of FAD-pyrophosphorylase and FAD-hydrolase (nucleotidepyrophosphatase) was studied in extracts of Pichia guilliermondii ATCC 9058 capable of riboflavin over-production. The specific activity of the enzymes was highest at the logarithmic growth phase (2.6 and 3.8 mcmoles of FAD per 1 min per 1 mg of protein X10(-5), respectively), and did not increase upon the induction of riboflavin overproduction. A decrease in the content of hemin compounds and a low content of flavins in the cells of Pichia guilliermondii mutants had no considerable effect on the activity of the two enzymes. When the yeast was cultivated on a medium containing hexadecane, an increase in the content of FAD in the cells was not accompanied with a rise in the activity of FAD-pyrophosphorylase. The activity of the enzyme did not change when succinate and lactate, the substrates of FAD-containing enzymes, were used as the source of carbon. The activity of FAD-pyrophosphorylase increased only when iron-deficient cells of the yeast were grown or incubated on a medium containing glycine; this stimulation was inhibited by cycloheximide.

[Increase in yeast and bacterial sensitivity to inhibitors and riboflavin as affected by high sulfate and phosphate concentrations]. / Uvelichenie chuvstvitel'nosti drozhzhei i bakterii k nekotorym ingibitoram i riboflavinu pod vozdeistviem vysokikh kontsentratsii sul'fata i fosfata.

Cultivation of the yeast Pichia guilliermondii in a medium with a high content of sulfate or phosphate ions (0.6 M and higher) increased its susceptibility to actinomycin D and 7-methyl-8-trifluoromethyl 10-(1'-D-ribityl)isoalloxazin, and analog of riboflavin, and decreased the requirement of the riboflavin-dependent mutant P7 in exogenous vitamin B2. The protoplasts of the yeast were also very susceptible to actinomycin D when they were incubated in a medium with a high sulfate concentration. Sulfate and phosphate ions elevated the susceptibility to actinomycin D in the following yeasts, apart from P. guilliermondii: Pichia pinus, Saccharomyces cerevisiae, Torulopsis candida, hansenula polymorpha, Schwanniomyces occidentalis, Candida utilis and Candida tropicalis. The growth of Escherichia coli was also very susceptible to actinomycin D when the bacterium was cultivated in medium with an elevated phosphate concentration (0.2 M). High phosphate or sulfate concentrations can be used in experiments aimed at studying the effect of transcription inhibitors (actinomycin D, 8-hydroxyquinoline) on the induction of alpha-glucosidase in P. guilliermondii.