Biosynthesis of xanthurenic acid 8-O-beta-D-glucoside in Drosophila. Characterization of the xanthurenic acid:UDP-glucosyltransferase activity

Xanthurenic acid 8-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila. From 3-hydroxykynurenine, two biosynthetic pathways can be envisaged, one via xanthurenic acid, and another via 3-O-glucoside of 3-hydroxykynurenine. In this report evidence is presented to show that the synthesis takes place via xanthurenic acid. (a) We have demonstrated that the Drosophila melanogaster vermilion purple mutant (unable to synthesize 3-hydroxykynurenine) synthesizes xanthurenic acid 8-glucoside when fed with xanthurenic acid; and (b) the activities required for its synthesis via xanthurenic acid have been found (3-hydroxykynurenine transaminase and xanthurenic acid:UDP-glucosyltransferase). This is the first time that a UDP-glucosyltransferase activity that utilizes xanthurenic acid has been demonstrated. The enzyme in crude extracts from Drosophila sordidula shows the following characteristics. (a) It has optimal activity at 35 degrees C at pH 7.1 (in buffer Tris-HCl), and in the presence of a divalent cation (Mg2+ or Mn2+); (b) the activity is inhibited by xanthurenic acid (above 1.5 mM), UDP, D-gluconic acid 1,5-lactone, and Triton X-100; (c) it is localized in both the microsomal and the soluble fractions; (d) the specific activity is two times higher in heads than in bodies; and (e) the activity is enhanced in flies fed with phenobarbital.     

The gametocyte-activating factor xanthurenic acid stimulates an increase in membrane-associated guanylyl cyclase activity in the human malaria parasite Plasmodium falciparum

Sex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg(2+)/Mn(2+) but is inhibited by Ca(2+). Significantly, this activity is increased on addition of xanthurenic acid. In contrast, a xanthurenic acid precursor (3-hydroxykynurenine), which is not an inducer of exflagellation, does not induce this guanylyl cyclase activity. These results therefore suggest that xanthurenic acid-induced exflagellation may be mediated by activation of the parasite cGMP signalling pathway.     

Xanthommatin biosynthesis in wild-type and mutant strains of the Australian sheep blowfly Lucilia cuprina

The synthesis of eye pigments has been studied in the seven eye color mutants of the Australian sheep blowfly, Lucilia cuprina. Six appear to be affected primarily in the synthesis of xanthommatin. In wild type, the onset of xanthommatin biosynthesis occurs midway through metamorphosis. Developmental patterns of accumulation of the xanthommatin precursors tryptophan, kynurenine, and 3-hydroxykynurenine have also been established for wild type. By determining the levels of these precursors in late pupae of the mutants, it has been shown that the mutant yellowish accumulates excess tryptophan and the mutant yellow accumulates excess kynurenine. The implications of these results—that yellowish lacks tryptophan oxygenase, thus failing to convert tryptophan to kynurenine, and that yellow lacks kynurenine hydroxylase (blocked in the conversion of kynurenine to 3-hydroxykynurenine)—have been confirmed. This has involved in vitro assays of tryphophan oxygenase and precursor feeding experiments. The precursor accumulation patterns are less clear for the other mutants.     

Xanthurenic acid provokes formation of unfolded proteins in endoplasmic reticulum of the lens epithelial cells

The role of xanthurenic acid in a cell is unknown, but it is suspected to provoke several diseases. This study shows that accumulation of xanthurenic acid in the lens epithelial cells leads to an overexpression of endoplasmic reticulum (ER) resident stress chaperones proteins, glucose-regulated protein (Grp94), and calreticulin. Both chaperones proteins are overexpressed in the presence of unfolded proteins. A formation of the unfolded protein in the presence of xanthurenic acid may take place due to covalent binding of xanthurenic acid to protein. Grp94 is responsible for scavenging of the unfolded proteins. The results suggest that Grp94 scavenged xanthurenic acid-modified proteins, and for this reason become preferentially yellow-stained in the presence of yellow xanthurenic acid. Such a modified Grp94 is weakly recognized by anti-Grp94 antibody. An end point of the xanthurenic acid accumulation in the cell is the cell death. In conclusion xanthurenic acid can lead to cell pathology.     

Oxidation of 3-hydroxykynurenine to produce xanthommatin for eye pigmentation: a major branch pathway of tryptophan catabolism during pupal development in the yellow fever mosquito, Aedes aegypti.

This study concerns the metabolic pathways of 3-hydroxykynurenine in Aedes aegypti mosquitoes during development with emphasis on its oxidation pathway to produce xanthommatin during eye pigmentation. Oxidation of tryptophan to 3-hydroxykynurenine is the major pathway of tryptophan catabolism in Aedes aegypti, but 3-hydroxykynurenine oxidizes easily under physiological conditions, which stimulate the production of reactive oxygen species. Our data show that in Aedes aegypti, the chemically reactive 3-hydroxykynurenine is converted to the chemically stable xanthurenic acid by a transaminase-catalyzed reaction during larval development, while 3-hydroxykynurenine is transported to the compound eyes for eye pigmentation during pupal development. Our data suggest that (1) the transamination pathway of 3-hydroxykynurenine is down-regulated during the pupal development, (2) 3-hydroxykynurenine produced in other body tissues is actively transported to the compound eyes during the pupal stage, (3) the compound eye is the place where ommochromes are produced, and (4) formation of ommochromes results from nonenzymatic oxidation of 3-hydroxykynurenine in the compound eyes.     

Distribution of xanthurenic acid glucoside in species of the genus Drosophila

Xanthurenic acid 8-O-β-d-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila melanogaster, that is accumulated in some eye-color mutants. Since this compound had only been found in this species, we have analyzed 29 other Drosophila species for the occurrence of this compound using cellulose thin-layer chromatography. Xanthurenic acid glucoside was detected in 10 of them (8 of them belong to the Sophophora subgenus). In these species xanthurenic acid glucoside, as well as other metabolites of the final steps of the dihydroxanthommatin pathway (3-hydroxykynurenine, xanthurenic acid and dihydroxanthommatin) were quantitated in order to gain a better understanding of the relationships of the metabolites of this pathway.     

Functions of the white and topaz loci of Lucilia cuprina in the production of the eye pigment xanthommatin

The white and topaz eye color mutants of L. cuprina are defective in the production of the brown screening pigment xanthommatin. Both white and topaz mutants were found to be unable to accumulate xanthommatin precursors in the larval malpighian tubules, correlating with their reduced early pupal level of this metabolite. In addition, white mutants showed reduced rates of accumulation of kynurenine and 3-hydroxykynurenine in the adult eyes. Another mutant strain, grape, was also defective in its ability to accumulate these xanthommatin precursors in the eyes, although accumulation was normal in the larval tubules. In contrast, the topaz mutants were found to be normal in eye accumulation, although tubule accumulation was markedly abnormal. These properties of the white and topaz mutants of L. cuprina are compared with those of the white and scarlet mutants of D. melanogaster, and it seems likely that in the two species these genes are involved with the uptake or storage of xanthommatin precursors in specific tissues.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee.     

Terminal Synthesis of Xanthommatin in DROSOPHILA MELANOGASTER. III. Mutational Pleiotropy and Pigment Granule Association of Phenoxazinone Synthetase

Phenoxazinone synthetase, which catalyzes the condensation of 3-hydroxykynurenine to xanthommatin, the brown eye pigment of Drosophila, is shown to exist in association with a particle which resembles the cytologically defined Type I pigment granule. Several classical eye color mutants (v, cn, st, ltd, cd, w), including two which effect other enzymes in the xanthommatin pathway (v, cn), have low levels of phenoxazinone synthetase activity and disrupt the normal association of the enzyme with the pigment granule. A model is proposed depicting several structural and enzymatic interrelationships involved in the developmental control of xanthommatin synthesis in Drosophila.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

A biochemical study of the scarlet eye-color mutant of Drosophila melanogaster

3-Hydroxykynurenine is virtually absent from st larvae but accumulates during adult development in the puparium. Over the period of adult emergence, the accumulated 3-hydroxykynurenine is excreted so that st adults contain none. Larvae of st fed on tryptophan-C ¹⁴ medium produce labeled 3-hydroxykynurenine, at a reduced rate, perhaps, compared to wild type. Xanthurenic acid levels in st pupae are similar to those in wild type. Thus the failure of st larvae to accumulate 3-hydroxykynurenine does not seem to be due either to an inability to synthesize this compound or to an excessive rate of its conversion to xanthurenic acid. Rather, it appears that the mechanism of 3-hydroxykynurenine storage during larval life is defective, so that this compound is excreted at an abnormally high rate. The inability of the pigment cells of the eyes of st to synthesize xanthommatin may result from a similar defect in their ability to take up or store 3-hydroxykynurenine.     

Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster

Several points of biochemical similarity between white and scarlet mutants suggest that both are defective in the transport of xanthommatin precursors. In both, accumulation of 3-hydroxykynurenine is negligible during larval life and occurs at only a slow rate during adult development. Larvae of both mutants also excrete 3H-3-hydroxykynurenine and 3H-kynurenine rapidly, which probably accounts for the normal levels of kynurenine during larval life. 3-Hydroxykynurenine levels are abnormal in all white mutants which were studied, although in two alleles which are strongly pigmented (w(sat) and w(col)) accumulation is enhanced rather than diminished. In w(a), larval accumulation is normal but accumulation during adult development is greatly diminished, suggesting that this mutation has a tissue-specific effect. Similar levels were found in zeste females. Of the 11 other eye color mutants tested, abnormal levels of 3-hydroxykynurenine were found in eight. In four of these (claret, light, lightoid, and pink), larval accumulation is negligible, suggesting that these have defects in the kynurenine transport system like scarlet and white. In three others, however (brown, karmoisin, and rosy), accumulation during larval life is enhanced. In cardinal accumulation is normal during larval life but is excessive during adult development. This evidence supports the suggestion that the cd mutation blocks the final step of xanthommatin synthesis.     

The ommochrome biosynthetic pathway in Drosophila melanogaster: The head particulate phenoxazinone synthase and the developmental onset of xanthommatin synthesis

Particulate fractions from the heads of Drosophila melanogaster catalyze the conversion of o-aminophenols to phenoxazinones. This particulate enzyme is stimulated by Mn²⁺. It has a number of features which distinguish it clearly from the Mn²⁺-dependent activity found in the soluble fraction. The particulate enzyme has a characteristic developmental pattern, showing a marked increase in activity at about the time of onset of xanthommatin synthesis. In addition, it is much reduced in activity in a number of xanthommatin-deficient mutants (v, cn, st, cd, and w). We believe that the head particulate enzyme is involved in xanthommatin biosynthesis and that the developmental onset of synthesis of this pigment is brought about by the synthesis or activation of this enzyme.     

Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid

Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH₄), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH₄ synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH₄ biosynthesis and BH₄-dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.     

Isolation and biochemical analysis of a temperature-sensitive scarlet eye color mutant of Drosophila melanogaster

Six new EMS-induced scarlet mutants were selected. Four of these were partially pigmented, with xanthommatin levels ranging from 12% to 45% of normal. In one (st ^754ts), pigment production was temperature sensitive; the level of xanthommatin changed from less than 10% of normal at 29 C to more than 70% at 18 C. In all of the new mutants tested, the level of early pupal 3-hydroxykynurenine was as low as low as that in st ¹. Thus reduced larval accumulation of this metabolite also appears to be a characteristic feature of scarlet mutants. Temperature-pulse and temperature-shift experiments were carried out with st ^754ts to determine the temperature-sensitive period for the scarlet gene during development. The major sensitive period commenced prior to the onset of pigmentation and was over before adult emergence. Thus the initiation of xanthommatin synthesis is not brought about by the activation of the scarlet gene. In similar experiments carried out with a temperature-sensitive white mutant (w ^bl), a similar temperature-sensitive period was obtained.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee and also by Grant GB 27599 from The National Science Foundation to Professor M. M. Green.     

Prooxidant action of xanthurenic acid and quinoline compounds: Role of transition metals in the generation of reactive oxygen species and enhanced formation of 8-hydroxy-2′-deoxyguanosine in DNA†

Xanthurenic acid, a product of tryptophan–NAD pathway, and quinoline compounds produced reactive oxygen species as a complex with iron. Aconitase, the most sensitive enzyme to oxidative stress was inactivated effectively by xanthurenic acid and to a lesser extent by 8-quinolinol in the presence of ferrous sulfate. The inactivation of aconitase was iron-dependent, and was prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that reduced iron bound to xanthurenic acid or 8-quinolinol can activate oxygen molecule to form superoxide radical. However, kynurenic acid and quinaldic acid without 8-hydroxyl group did not produce reactive oxygen species. Of the quinoline compounds tested, xanthurenic acid and 8-quinolinol with 8-hydroxyl group stimulated the autooxidation of ferrous ion, but kynurenic acid and quinaldic acid did not affect the oxidation of ferrous ion. Hydroxyl group at 8-positions of quinoline compounds was essential for the binding of iron causing the generation of reactive oxygen species. 8-Quinolinol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA, suggesting the quinolinol/copper-dependent stimulation hydroxyl radical formation. Xanthurenic acid and 8-quinolinol as the metal–chelate complexes can show various cytotoxic effects by generating reactive oxygen species through the ferrous or cuprous ion-dependent activation of oxygen molecule. † This paper is dedicated to centennial of the birthday of the late Professor Emeritus Yahito Kotake, a pioneer of the xanthurenic acid research.     

Metabolism and Biosynthesis of Cytokinins in Tobacco Crown Gall Cells

The metabolism of ribosylzeatin (RZ) was studied using tobaccocrown gall cells which produce RZ as one of the major endogenouscytokinins. When [8-¹⁴C]RZ was fed to the cells, it was convertedinto its phosphate (which was rigorously determined to be the5'-monophosphate), RZ-O-glucoside, inosine (or its phosphate),adenosine and adenosine-O-glucoside. When [8-¹⁴C]N⁶-(²-isopentenyl)adenosine(i⁶Ado), a probable precursor of RZ, was fed to the cells, itwas converted into (i⁶Ado)-O-glucoside, inosine (or its phosphate),adenosine, adenosine-O-glucoside and adenosine phosphate, butno incorporation of radioactivity into RZ was observed. Thepresent study led to the following conclusions: i) i⁶Ado isnot a precursor of RZ in the cells, ii) both deaminase and cytokininoxidase are involved in the catabolism of cytokinin, and iii)the metabolism of RZ is quite different from that of i⁶Ado. (Received December 24, 1985; Accepted April 1, 1986)     

Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1

Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The K(m) of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The K(m) of bmST1 for xanthurenic acid is low, in the μM range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm.     

Terminal synthesis of xanthommatin in drosophila melanogaster. IV. Enzymatic and nonenzymatic catalysis

Nonenzymatic and enzymatic catalysis of the oxidation of 3-hydroxykynurenine (and 3-hydroxyanthranilic acid) has been studied and characterized in Drosophila extracts, clearing up some of the confusion surrounding the synthesis of the brown eye pigment, xanthommatin. The genetic basis of the terminal steps in pigment synthesis remains obscure, since all mutants tested have full synthetase activity.     

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.     

The Arabidopsis PLEIOTROPIC DRUG RESISTANCE8/ABCG36 ATP Binding Cassette Transporter Modulates Sensitivity to the Auxin Precursor Indole-3-Butyric Acid

Plants have developed numerous mechanisms to store hormones in inactive but readily available states, enabling rapid responses to environmental changes. The phytohormone auxin has a number of storage precursors, including indole-3-butyric acid (IBA), which is apparently shortened to active indole-3-acetic acid (IAA) in peroxisomes by a process similar to fatty acid β-oxidation. Whereas metabolism of auxin precursors is beginning to be understood, the biological significance of the various precursors is virtually unknown. We identified an Arabidopsis thaliana mutant that specifically restores IBA, but not IAA, responsiveness to auxin signaling mutants. This mutant is defective in PLEIOTROPIC DRUG RESISTANCE8 (PDR8)/PENETRATION3/ABCG36, a plasma membrane–localized ATP binding cassette transporter that has established roles in pathogen responses and cadmium transport. We found that pdr8 mutants display defects in efflux of the auxin precursor IBA and developmental defects in root hair and cotyledon expansion that reveal previously unknown roles for IBA-derived IAA in plant growth and development. Our results are consistent with the possibility that limiting accumulation of the IAA precursor IBA via PDR8-promoted efflux contributes to auxin homeostasis.