Correlation of guanosine triphosphate cyclohydrolase activity and the synthesis of pterins in Drosophila melanogaster

The enzyme guanosine triphosphate cyclohydrolase (GTP cyclohydrolase), which in bacteria is known to be the first enzyme in the biosynthetic pathway for the synthesis of pteridines, has been discovered in extracts of Drosophila melanogaster. Most of the enzyme (80%) is located in the head of the adult fly. An analysis of enzyme activity during development in Drosophila has revealed the presence of a relatively small peak of activity at pupariation and a much larger peak that appears at about the time of eclosion. Enzyme activity declines rapidly as the fly ages. Analyses for the production of the typical pteridine pigments of Drosophila have indicated that the small peak of GTP cyclohydrolase activity evident at pupariation coincides with the appearance of isoxanthopterin, sepiapterin, and pterin, and the larger peak at eclosion roughly corresponds to the accumulation of drosopterin as well as to the appearance in larger amounts of pterin and sepiapterin. These observations strongly suggest that in Drosophila, like bacteria, GTP cyclohydrolase is involved in the biosynthesis of pteridines. Analyses of a variety of zeste mutants of Drosophila melanogaster have shown that these mutants all contain GTP cyclohydrolase equal approximately to the amount found in the wild-type fly. These observations do not support the suggestions made by Rasmusson et al. (1973) that zeste is the structural locus for GTP cyclohydrolase.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation inDrosophila melanogaster

The relationship between high dietary levels of aromatic amino acid and regulation of pteridines inDrosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected byl-phenylalanine,l-tryptophan, andl-tyrosine in decreasing order and thed-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the threel-amino acids. Pterin was regularly increased, and isoxanthopterin decreased.l-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study;l-tryptophan also caused dihydrobiopterin to increase butl-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine,Pu ² andHn ^r3. These two mutants, along withpr ^c4 cn/pr ^m2b cn, were shown to be very sensitive to dietaryl-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.This work was sponsored in part by a grant from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Biosynthesis of "drosopterins" by an enzyme system from Drosophila melanogaster.

The red eye pigment of Drosophila melanogaster consists of six complex pteridines known as neodrosopterin, drosopterin, isodrosopterin, fraction e, and aurodrosopterins (2); these pigments are greatly reduced in the purple mutant. Conditions for biosynthesis of these "drosopterins" are described and compared with those for the synthesis of sepiapterin. The enzymes are contained in a soluble, pteridine-free extract obtained between 40 and 60% saturated ammonium sulfate. The results indicate that sepiapterin synthase consists of two enzymes, the first of which provides a precursor for "drosopterin" biosynthesis. The evidence is (1) the purple mutant, low in accumulated sepiapterin and "drosopterins", is known to have approximately 10% of the sepiapterin synthase activity of wild type; (2) unlabeled sepiapterin does not cause isotope dilution of "drosopterin" synthesis; (3) the 600g pellet prepared from a wild-type head homogenate contains "drosopterin" synthesizing activity and no sepiapterin synthase, yet a heat-labile factor in this fraction stimulates sepiapterin synthesis in the 100000g supernatant of wild-type or pr flies; (4) sepiapterin and "drosopterin" syntheses require Mg2+; (5) sepiapterin synthesis is stimulated by NADPH; "drosopterin" synthesis responds to either NADPH or NADH. Although "drosopterins" are complex pteridine-type pigments, we have demonstrated their biosynthesis by soluble enzymes. This allows us to consider investigation into the mechanism by which the amounts of these pigments are regulated.     

Genetic and biochemical analysis of brown eye mutation in Drosophila nasuta nasuta and Drosophila nasuta albomicans

By analyzing the progeny of crosses involving brown eye mutants and the wild types in two members of Drosophila nasuta subgroup namely D. n. nasuta and D. n. albomicans we could show that the mutant gene is recessive, located in the chromosome 2 and the alleles of this gene are present at different loci. A study of fitness in the eye color mutants in comparison with the wild types revealed that D. n. nasuta mutant has higher viability at both 25 ± 1°C and ambient temperatures; while D. n. albomicans mutant has faster rate of development only at 25 ± 1°C. Quantitative analysis of eye pigments in the mutants revealed that there is biosynthesis of both pteridines and xanthommatins unlike in bw/bw of D. melanogaster, where only xanthommatins are synthesized. In both the species, the pteridine quantities in mutants are similar; whereas xanthommatin quantity in is 10 times higher than that of . Further, the F₁ progeny of intraspecific crosses (wild type X mutant) are found to have high amounts of pteridine, even when compared with parental wild type. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of Pteridines in the Body Coloration of the Isopod Armadillidium vulgare

The pteridine content was measured as a function of age in Armadillidium vulgare, and the fine structure of the pteridine-containing granules in the integument was examined in relation to pteridine content. Yellow chromatophores are an essential component of the cream-markings, which are a defining feature of the female A. vulgare. Four kinds of pteridines in the integument including a yellow pigment (sepiapterin) were determined by HPLC. The body color of the red phenotype of A. vulgare varies from dark red to yellowish red and was formerly thought to be due to the quality and quantity of ommochrome pigment. Our analysis of the pteridine content in the integument of this phenotype revealed a significant change in sepiapterin content per body weight with age. Sepiapterin content per body weight decreased gradually with age, while that of biopterin tended to increase with age. Ultrastructural observations of the pigment granules in the yellow chromatophores revealed a corresponding change in the fine structure of pigment granules. In the older adults, some of the electron-dense fibrous materials in the pteridine-containing granules was concentrically arranged, and in the younger adults, most of pteridine-containing granules were electron-lucent. The role of pteridine quality in determining the structure of pteridine-containing granules is discussed.     

Nickel complexes of sepiapterin and 6-acetyldihydrohomopterin, a pyrimidodiazepine from Drosophila

The nonfluorescent pyrimidodiazepine in Drosophila melanogaster 6-acetyldihydrohomopterin (H₂Ahp) was studied using ultraviolet and infrared spectroscopy. The H₂Ahp was unstable in 3% NH₄Cl whereas a related pteridine sepiapterin was stable. Since Ni²⁺ stabilized H₂Ahp completely, the structure of the H₂Ahp · Ni complex was examined. Among 15 pterins, including sepiapterin, the spectral properties in the presence of Ni²⁺ reflect the pK_a's and the reactive group on the side chain but for H₂Ahp the spectral properties are rather different from the pteridines and they indicate that the seven-membered ring seemed to have the predominant influence. The Ni²⁺ comples of H₂Ahp resulted in a shift in the absorption maximum from 383 to 436 nm. The corresponding spectral shift of the pteridines due to Ni²⁺ was much less. From the infrared spectra of H₂Ahp and sepiapterin in the presence and absence of Ni²⁺, the sites of interaction of Ni²⁺ with H₂Ahp were shown to be the phenolic oxygen and N5 in the ring. In the absence of Ni²⁺ an internal hydrogen bond in sepiapterin was indicated that may involve the carbonyl oxygen and the secondary alcoholic oxygen on the side chain. Other metal ions were tested (Cd²⁺, Zn²⁺) but were not as effective as Ni²⁺ in stabilizing H₂Ahp.     

Developmental changes of sepiapterin synthase activity associated with a variegated purple gene in Drosophila melanogaster

A variegated position effect on the autonomous gene, purple, has been studied enzymologically in Drosophila melanogaster. Sepiapterin synthase, the enzyme system associated with pr ⁺, was examined for activity in different developmental stages of the fly. The results indicate that T(Y:2) pr ^c5, cn/pr^c4 cn flies (flies in which pr ⁺ has been translocated and which exhibit variegation) have a reduced amount of enzyme activity as compared with both Oregon-R and pr ¹ flies. This reduction in activity was not found in larval stages, which suggests that the inactivation process probably occurs in late larval or early pupal stages. The phenotype of the variegated adult has white eyes with red-colored spots and patches where drosopterins occur. The phenotype of the fly carrying the translocation is modified by the presence of additional Y chromosomes. This extends the observation from other systems that extra heterochromatin acts to suppress the variegated position effect. The advantages of studying the variegation by measuring enzyme activity, as well as the phenotypic expression, are several; for example, the developmental time at which variegation occurs may be estimated even though drosopterin synthesis is not occurring.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the Department of Energy under Contract No. W-7405-eng-26.     

Uptake and incorporation in pteridines of externally supplied GTP in normal and pigment-deficient eyes ofDrosophila melanogaster

Some aspects of the synthesis of drosopterins in the eyes ofDrosophila melanogaster have been studied in flies with different levels ofwhite gene expression. The activity of GTP cyclohydrolase was found to differ between wild-type and yellow-eyed mutantsin vivo but notin vitro. To elucidate the uptake of substrate, we measured the removal of labeled GTP from the incubation medium by excised pupal eyes and followed the subsequent fate of this label. It was found that GTP was dephosphorylated to guanosine extracellularly before label was taken up by the eye tissue. The uptake was much lower in yellow and white eyes than in wild-type eyes, and in the latter, a considerable part of the label was present in pteridine compounds. The strain differences in the uptake of label seem to be due to different rates of intracellular utilization of guanine derivatives in pteridine synthesis. We suggest that this is caused by a hampered transport of precursor (possibly GTP) in white andzeste eyes through the membrane of red pigment granules.This project was sponsored by the Swedish Natural Science Research Council.     

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.     

Yellow Marking and Pteridine Contents in the Integument of Albino Armadillidium vulgare

The albino mutant strain in the woodlice, Armadillidium vulgare, was investigated with respect to the yellow patterns on the dorsal integument. Pigment cells were observed with electron microscope in order to determine the cell types of yellow markings. Quantitative analyses of pteridines in the albino were carried out by HPLC. The result indicated that the albino integument contain sepiapterin, biopterin, pterin, isoxanthopterin as in the wild type and the red mutant strain. The total amount of the four pteridines in the albino was about half as much as that in the red phenotype for both males and females, respectively. Males and females showed almost the same totals and ratios of the four pteridines in the albino and red phenotypes. Therefore, pteridine contents in both phenotypes of A. vulgare may not be related to the activity of androgenic gland hormone. Yellow chromatophores of the albino and red phenotypes were morphologically identical, emitting a yellow fluorescence. These cells contained numerous electron-lucent pigment organelles which were similar to pteridine granules of the wild type.     

A Genetic Analysis of the Pteridine Biosynthetic Enzyme, Guanosine Triphosphate Cyclohydrolase, in DROSOPHILA MELANOGASTER

Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues.     

Phenol oxidase activity and the Lozenge locus of Drosophila melanogaster

We have found that the phenol oxidase activity in 50-hr Drosophila melanogaster pupae is much greater than that of adult flies. The mutants lz and lz ^g have all of the phenol oxidase components present in wild type, whereas the mutant tyr-1 has all of the wild-type components but the activity of each component is greatly reduced in comparison with wild-type activity. The newly discovered lozenge allele, lz ^rfg, lacks all phenol oxidase activity.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish

Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. Here, we investigate the pigment phenotype and, from this, provide evidence for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis.     

Isolation and characterization of pteridines from heads of Drosophila melanogaster by a modified thin-layer chromatography procedure

An improved thin-layer chromatography technique is described for the separation of fluorescent compounds found in extracts of heads of Drosophila melanogaster. Eighteen to twenty fluorescent spots are resolved, two of which are xanthurenic acid and 3-hydroxykynurenine, and the remaining spots are presumably pteridines. Of these, nine have been identified and quantitated directly on the chromatograms with a fluorometer. One of the spots present on the chromatogram apparently has not been described previous to this work. Characteristics of this substance, termed quench spot, are presented, several of which indicate that it may be a pteridine or pteridine derivative.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Mechanism of suppression inDrosophila: Regulation of tryptophan oxygenase by thesu(s) + allele

The suppressor gene,su(s)², inDrosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of thesu(s)⁺ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removingsu(s)⁺ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts fromsu(s)⁺ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or –20°C or by boiling, whereas the wild-type enzyme is not affected by extracts ofsu(s)⁺ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy ofsu(s)⁺ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of thesu(s)⁺ allele. How thesu(s)⁺ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.This work was supported in part by a grant from the KOSEF, Korea Science and Engineering Foundation, and the National Science Foundation under the U.S.-East Asia Cooperative Science Program as well as the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.