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.     

The red mutations impair the regulation of flavinogenesis and metal homeostas in yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib+ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib- phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib+ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, and hit1) and six new loci designated red1-red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I), and to H2O2. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

The cellular origin and the control of neopterin release associated with immune stimulation was studied in cell cultures. Using purified human mononuclear cells, the intracellular change in concentrations of GTP and pterins was measured under various kinds of stimulation. Three enzymes involved in tetrahydrobiopterin biosynthesis, i.e. GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, were also determined. Human macrophages stimulated with culture supernatant from activated T-lymphocytes were the main producers of neopterin. In these cells, GTP cyclohydrolase I activity was elevated due to high GTP levels and therefore neopterin accumulated. Human macrophages lack 6-pyruvoyl tetrahydropterin synthase activity. Exogenous tetrahydrobiopterin added to the culture medium of stimulated T cells and macrophages suppressed the elevation of GTP cyclohydrolase I activity and neopterin concentration, but not the elevation of intracellular GTP. Stimulation of macrophages with recombinant human interferon-gamma and neutralization of the effect of T cell supernatants by addition of a monoclonal antibody specific for human interferon-gamma showed that immune interferon induced the alterations in GTP cyclohydrolase I activity and neopterin concentration. In the human macrophage line U-937 and in the leukemia line HL-60, no GTP cyclohydrolase I activity or intracellular pterins were detected, but high levels of GTP. In mouse mononuclear cells, no neopterin was detected, but biopterin and pterin. After stimulation, biopterin was elevated in the same way as neopterin in human mononuclear cells. This is explained by the different regulation of the rate-limiting steps of tetrahydrobiopterin biosynthesis in man and in mouse. These results suggest that neopterin is an unspecific marker for the activation of the cellular immune system.     

First reaction of riboflavin biosynthesis —Catalysis by a guanosine triphosphate cyclohydrolase from yeast

An enzyme that uses GTP as a substrate for the formation of formate and a 2.4.5-triamino-6-hydroxypyrimidine derivative has been determined in the riboflavin overproducing yeast Pichia guilliermondii. In rib 1 mutants this enzyme is absent. This implies an involvement of the enzyme in the riboflavin biosynthesis and indicates that GTP is a purine precursor of riboflavin. Some properties of the GTP cyclohydrolase (substrate specificity, pH and temperature optima, activators and inhibitors, molecular weight, stability) were studied using a partly purified enzyme preparation. Cells grown under riboflavin overproducing conditions (iron deficiency) have 20–40-fold increased enzyme activity as compared with non-overproducing cultures (supplemented with iron).Non-Standard Abbreviations RF riboflavin - DHRiboyslAP £ 2.5-diamino-6-hydroxy-4-(1-d-ribosylamino)pyrimidine-5-phosphate - DHRibitylAP 2.5-diamino-6-hydroxy-4-(1-ribitylamino)pyrimidine - neopterin 6-(trihydroxypropyl)pterin - DMP 6.7-dimethylpterin - Fe iron deficient condition - +Fe supplemented with iron     

Control of drosopterin synthesis in Drosophila melanogaster: Mutants showing an altered pattern of GTP cyclohydrolase activity during development

The reaction catalyzed by GTP cyclohydrolase is the first unique step of pteridine biosynthesis in Drosophila melanogaster and is therefore likely to be an important control point. GTP cyclohydrolase activity varies during development, showing two distinct peaks of activity—one at pupariation and a much larger peak at emergence. Most of the early pupal enzyme is located in the body region, whereas in late pupal and early adult life most of the activity is found in the head. Mixing experiments indicate that developmental changes in activity are not due to changes in the level of a direct effector of GTP cyclohydrolase. The mutants raspberry and prune show an increased GTP cyclohydrolase activity at pupariation relative to wild type, but a decreased enzyme activity at emergence. The changes in GTP cyclohydrolase activity are reflected in changes in pteridine levels in these mutants. Several lines of evidence suggest that neither locus is the structural gene for GTP cyclohydrolase. The raspberry and prune gene products may play a specific role in regulating GTP cyclohydrolase activity during development.This work was supported by a grant from the Australian Research Grants Committee D2 75/15248.     

Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

The red Mutations Impair the Regulation of Flavinogenesis and Metal Homeostasis in Yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib⁺ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib^– phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib⁺ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, andhit1) and six new loci designated red1–red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I)) and to H₂O₂. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.     

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.     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Description of a Riboflavin Biosynthetic Gene Variant Prevalent in the Phylum Proteobacteria

Riboflavin (vitamin B₂) is the precursor of flavin mononucleotide and flavin adenine dinucleotide, which are cofactors essential for a host of intracellular redox reactions. Microorganisms synthesize flavins de novo to fulfill nutritional requirements, but it is becoming increasingly clear that flavins play a wider role in cellular physiology than was previously appreciated. Flavins mediate diverse processes beyond the cytoplasmic membrane, including iron acquisition, extracellular respiration, and interspecies interactions. While investigating the regulation of flavin electron shuttle biosynthesis in the Gram-negative gammaproteobacterium Shewanella oneidensis, we discovered that a riboflavin biosynthetic gene (ribBA) annotated as encoding a bifunctional 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase/GTP cyclohydrolase II does not possess both functions. The novel gene, renamed ribBX here, encodes an amino-terminal DHBP synthase domain. The carboxy-terminal end of RibBX not only lacks GTP cyclohydrolase II activity but also has evolved a different function altogether in S. oneidensis, regulating the activity of the DHBP synthase domain. Phylogenetic analysis revealed that the misannotation of ribBX as ribBA is rampant throughout the phylum Proteobacteria (40% of 2,173 annotated ribBA genes) and that ribBX emerged early in the evolution of this group of microorganisms. We examined the functionality of representative ribBX genes from Beta-, Gamma-, and Epsilonproteobacteria and found that, consistent with sequence-based predictions, the encoded GTP cyclohydrolase II domains lack catalytic activity. The persistence of ribBX in the genomes of so many phylogenetically divergent bacterial species lends weight to the argument that ribBX has evolved a function which lends a selective advantage to the host.     

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.     

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.     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase

Summary The enzyme inositol-1-phosphate synthase is repressed at least 50-fold in wild type yeast grown in inositol-supplemented media. Mutants which synthesize this enzyme constitutively have been isolated using a selection procedure based on excretion of inositol into the growth medium by putative mutants. Biochemical analysis of one of the mutants (opi1-1) confirmed that the nature of the mutations is regulatory, and not in the structural gene for the enzyme. Immunoprecipitation of crude extracts with antibody directed against purified inositol-1-phosphate synthase showed that a protein which reacts with the antibody is present in the mutant grown under both repressing and derepressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed. Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and drepressing conditions. Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD. These analyses indicated that purified mutant synthase was similar to the wild type enzyme.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

GTP cyclohydrolase I is coinduced in hepatocytes stimulated to produce nitric oxide

GTP cyclohydrolase I is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide (NO) synthase. Here we show that GTP cyclohydrolase I mRNA was present in unstimulated hepatocytes and was up-regulated 2- to 3-fold concurrently with iNOS induction induced in vivo by LPS injection and in vitro by stimulation with LPS and inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta, and interferon-gamma. Hepatocyte GTP cyclohydrolase I enzyme activity increased 2-fold in vivo after LPS. This coinduction of GTP cyclohydrolase I resulted in increased total intracellular biopterin which supported induced NO synthesis. The addition of a GTP cyclohydrolase I inhibitor to the stimulated hepatocytes decreased intracellular biopterin levels and resulted in a decrease in NO production. The results show that GTP cyclohydrolase I is up-regulated by certain acute inflammatory conditions. Further, the results indicate that biopterin is essential as a cofactor for induced NO synthase activity in hepatocytes.