Molecular cloning of the GTP-cyclohydrolase structural gene RIB1 of Pichia guilliermondii involved in riboflavin biosynthesis.

The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P. guilliermondii rib1 mutant was isolated and sequenced.

[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.

[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.

[Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts].

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.

[Regulation of the activity and synthesis of enzymes participating in the formation of 6,7-dimethyl-8-ribityllumazine, a riboflavin precursor in yeast].

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.

[Supersynthesis of flavins in microorganisms and its molecular mechanism (review of the literature)].

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.

[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.

[The nature of the pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase participating in riboflavin biosynthesis in yeasts].

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.

[Flavin biogenesis in yeasts].

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.

[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.

[GTP-cyclohydrolases of microorganisms and their role in metabolism].

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.

[Changes in the enzyme activity of flavinogenesis in the process of culturing the fungus Eremothecium ashbyii].

The activity of enzymes involved in the beginning (GTP cyclohydrolase) and terminal steps (riboflavin synthase EC 2.5.1.

[Localization of the genes coding for GTP cyclohydrolase II and riboflavin synthase on the chromosome of 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.

[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.

[Flavinogenesis regulation in riboflavin-dependent Escherichia coli mutants].

355 Escherichia coli mutants requiring riboflavin (RF) for their growth were selected using N-methyl-N-nitro-N-nitrosoguanidine. The mutants were subdivided into four biochemical groups according to their capability to accumulate fluorescent pigments in the medium and their nature, the ability to grow in a medium containing either diacetyl or 6,7-dimethyl-8-ribityl lumazine (DMRL), and the activity of GTP cyclohydrolase II and RF synthase. The rate of RF accumulation by the parent strain of E.

[Effect of riboflavin analogs on Pichia guilliermondii growth].

The work was aimed at studying the biological activity of 32 structural riboflavin (RF) analogs substituted at positions 7, 8 and 10 as well as with a modified structure of the isoalloxazine cycle and the side D-ribityl chain. An RF-dependent mutant of the yeast Pichia guilliermondii MS1 was used as a test organism. The strain could grow in a medium without the vitamin in the presence of 2-thio-RF and analogs with esterified hydroxyls in the side chain, viz.

[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.

[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.

[Selection and mutant properties of Pichia guilliermondii yeasts with derepressed GTP cyclohydrolase, the enzyme of the 1st step in flavinogenesis].

A positive method is proposed for selecting Pichia guilliermondii mutants with derepressed GTP cyclohydrolase. Mutants with the incompletely blocked gene RIB2 were used as parent strains; these can grow in a medium without riboflavin (RF) only if the enzyme is derepressed as the result of iron deficiency in cells. Strains growing in a medium without RF at the optimal supply of cells with iron were selected as regulatory mutants.

[Activity of the enzyme of the 2d step of flavinogenesis, 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, in Pichia guilliermondii yeasts].

The activity of 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, an enzyme of the second step of flavinogenesis, was studied in the yeast Pichia guilliermondii using a new fluorimetric technique. The activity of the enzyme was found in extracts of the wild-type strain and the genotype rib mutants but not in extracts of the FM 1-4 (rib 2) mutant. Interallelic complementation was revealed in the gene rib 2.

[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.

[Increase in yeast and bacterial sensitivity to inhibitors and riboflavin as affected by high sulfate and phosphate concentrations].

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.

[Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide].

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.

[Overproduction of riboflavin in mutants of Pichia guilliermondii yeasts resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine].

Mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine were selected from the yeast Pichia guilliermondii, strain MS14-A10, possessing a multiple sensitivity to antibiotics and antimetabolites. A lot of such mutants displayed an elevated flavinogenic activity. The investigation of the properties of three mutants with the highest flavinogenic activity, viz.

[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.