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.

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.

[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.

[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.

[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.

[Mechanism of retroinhibition in e regulation of flavinogenesis in yeasts of genus Pichia]. / O mekhanizme retroingibirovaniia v reguliatsii flavinogeneza v drozhzhei roda 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.

[Product of second-stage riboflavin biosynthesis in Pichia guilliermondii]. / O produkte vtorogo étapa biosinteza riboflavina u Pichia guilliermondii.

Dialyzed extracts of the mutants of Pichia guilliermondii RG80 (rib3, his-) and RA39 (rib3, ade-) in which the third step of flavinogenesis was genetically blocked converted GTP into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate and 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine in the presence of NADPH2. Apparently, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate is a true precursor of riboflavin whereas 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine is formed upon its hydrolysis.

[Detection of phosphorylated pyrimidine precursors of riboflavin in yeasts]. / Obnaruzhenie fosforilirovannykh pripmidinovykh predshestvennikov riboflavina u drozhei.

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.

[Nature of riboflavin precursors in Pichia guilliermondi yeasts]. / Priroda predshestvennikov riboflavina u drozhzhei Pichia guilliermondii

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.

[Purification and properties of GTP-cyclohydrolase of the yeast Pichia guilliermondii]. / Ochistka i svoistva GTP-tsiklogidrolazy drozhzhei 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.

[Effect of iron, actinomycin D and cycloheximide on the GTP-cyclohydrolase synthesis in flavinogenic yeasts]. / Vliianie zheleza, aktinomitsina D i tsiklogeksimida na sintez GTP-tsiklogidrolazy u flavinogennykh drozhzhei.

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.

[Regulation of synthesis of GTP-cyclohydrolase participating in yeast falvinogenesis by iron]. / Reguliatsiia zhelezom sinteza GTF-tsiklogidrolazy, uchastvuiushchei vo flavinogeneze drozhzhei.

Pichia guilliermondii, Schwanniomyces occidentalis, Torulopsis candida and several riboflavin-dependent mutants of Torulopsis candida were grown in a medium with a low concentration of iron. In these conditions, the activity of GTP-cyclohydrolase which catalyzes the first step of flavinogenesis increases. The activity of the enzyme increases also when the cells of T. candida and P. guilliermondii with a high content of iron are incubated with alpha, alpha'-dipyridyl which induces overproduction of riboflavin; this action of alpha, alpha'-dipyridyl is eliminated by cycloheximide. Therefore, iron deficiency in the cells of these yeasts causes derepression of GTP-cyclohydrolase participating in riboflavin biosynthesis. The activity of the enzyme is inhibited by FAD but not by FMN and riboflavin.