Developmental regulation of tryptophan catabolism in Drosophila

The levels of tryptophan oxygenase and kynurenine formamidase present during Drosophila development were measured. The pattern of activity for both enzymes during development is bimodal. The activity of kynurenine formamidase exceeds that of tryptophan oxygenase by about 100 fold and maximal levels of tryptophan oxygenase are reached somewhat later than kynurenine formamidase. These observations indicate that tryptophan oxygenase is rate limiting in the catabolism of tryptophan. Activities were also measured in a number of eye color mutants. Some of these have elevated tryptophan oxygenase activity, as compared to wild type, in adults shortly after emergence. Assays of mixed extracts showed that the activity differences are not due to the presence of inhibitors or activators. Genetic analyses using two of the eye color mutants, ca and w, showed that the activity differences segregate with the eye color mutant loci. These data led us to hypothesize that tryptophan oxygenase activity is elevated in these mutants due to kynurenine accumulation. This hypothesis was confirmed by showing that tryptophan oxygenase activity in wild type animals is proportional to the concentration of tryptophan or kynurenine added to the media. Kynurenine accumulation in ca and w was show to be greater in the mutants, specifically at the time when the activity differences between these mutants and wild type were greatest. We have concluded from these studies that tryptophan oxygenase activity in Drosophila can be controlled by the concentration of kynurenine.     

Isolation and characterization of an hydroxykynureninase from homogenates of adult mouse liver

A kynureninase-type enzyme was isolated from adult mouse liver. With kynurenine as the substrate, this enzyme has a K_m of 300 μ$\text{M}$; when the substrate is hydroxykynurenine, the K_m is 6 μ$\text{M}$. We conclude that this enzyme is an hydroxykynureninase. No enzyme which we could characterize as a kynureninase was found in this preparation. This suggests that tryptophan metabolism in the mouse occurs primarily through pathways that use hydroxykynurenine rather than kynurenine. Preliminary studies indicate that the enzyme is inhibited by its reaction product, hydroxyanthranilate, which is an intermediate in the synthesis of NAD. Such control of the hydroxykynureninase reaction may be of physiological importance in regulating the synthesis of NAD and/or in preventing the accumulation of hydroxyanthranilate, a putative carcinogen.     

Purification and characterization of kynurenine formamidase activities from Streptomyces parvulus

Two forms of kynurenine formamidase (EC 3.5.1.9; aryl-formylamine amidohydrolase) are present in extracts of Streptomyces parvulus. The higher molecular weight enzyme (Mr = 42 000), kynurenine formamidase I, appears to be constitutive and is present at relatively constant but low levels in antibiotic producing and nonproducing cultures, whereas the synthesis of the lower molecular weight form (Mr = 25 000), kynurenine formamidase II, is initiated just prior to the onset of actinomycin formation. It is postulated (i) that kynurenine formamidase II catalyzes the second step in the pathway from tryptophan----actinocin, and (ii) that it is regulated specifically for the specialized function of actinomycin biosynthesis. The role of kynurenine formamidase I is unknown. Formamidase I and II activities were purified from extracts of S. parvulus and kinetic parameters of the two enzymes were determined. Although some of the properties of the two enzymes are quite similar (substrate specificities, Km values), some striking differences were noted (pH and temperature optima, molecular size, chromatographic properties, sensitivity to certain ions and chemicals). Mutant studies suggest that expression of the gene(s) coding for formamidase II activity play an essential role in regulating the formation of actinocin and, hence, antibiotic synthesis. Kynurenine formamidase activity was also found in a representative number of Streptomyces species and related organisms suggesting that the enzyme may function in the degradative metabolism of tryptophan by certain actinomycetes in nature.     

Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.     

Product induction in Pseudomonas acidovorans of a permease system which transports L-tryptophan

Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.     

Regulation of the synthesis of enzymes of tryptophan dissimilation in Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus dissimilates tryptophan via the -ketoadipate pathway. The first enzyme, tryptophan oxygenase (l-tryptophan: oxygen oxidoreductase; EC 1.13.1.12), is substrate-induced by tryptophan. The second two enzymes, formamidase (aryl-formylamine amidohydrolase; EC 3.5.1.9) and kynureninase (l-kynurenine hydrolase; EC 3.7.1.3), are induced by the next intermediate, kynurenine. The last enzyme specific to tryptophan dissimilation, anthranilate oxidase, is substrate induced. This inductive pattern is in marked contrast to the extensive coordinacy of enzyme synthesis characteristic of the remainder of the -ketoadipate pathway.     

A specific and sensitive fluorometric assay for tryptophan oxygenase

A spectrophotofluorometric assay was used to measure tryptophan oxygenase activity in several species. The fluorescent assay depends on the conversion of the product of the reaction, N-formyl-l-kynurenine, to anthranilate by means of the coupling enzymes kynurenine formamidase and kynureninase. These enzymes are easily obtained from l-tryptophan-induced N. crassa; and the product, anthranilate, is readily separated by organic extraction from other tryptophan catabolites and easily identified fluorometrically. With this assay, tryptophan oxygenase has been demonstrated in vitro for the first time in N. crassa.     

Control of the actinomycin biosynthetic pathway in and actinomycin resistance of Streptomyces spp.

Using actinomycin-producing and nonproducing strains of Streptomyces antibioticus, I studied several steps in the biosynthetic pathway of this antibiotic. Actinomycin-nonproducing strains derived after acriflavine or novobiocin treatment showed activity of kynurenine formamidase and phenoxazinone synthase as high as that of the parental strain, but these nonproducing strains failed to convert 4-methyl-3-hydroxy-anthranilic acid to actinomycin. In addition, accumulation of 4-methyl-3-hydroxyanthranilic acid (in the presence of D-valine) was not detected in the nonproducing isolates. Actinomycin-nonproducing strains derived after acriflavine treatment of Streptomyces parvulus showed a drastic decrease of resistance to the antibiotic. However these strains regained resistance after preincubation with a small amount of actinomycin D.     

Rates of sterol synthesis and hydroxymethylglutaryl coenzyme a reductase levels,and the effects of cholest-4-en-3-one on these parameters,in the livers of inbred strains of mice

Hereditary factors in inbred mouse strains affected the rate of sterol synthesis from acetate and the level of hydroxymethylglutaryl coenzyme A reductase in liver in two ways. During the forenoon, rates of sterol synthesis and levels of HMG-CoA reductase activity were two- to five-fold higher in C57L/J and DBA/2J mice than in mice of strains A/HeJ or SWR/J. Due to an apparent difference in the circadian cycle of the two strains, these differences between C57BL/6J and A/HeJ strains were not as great at 4:30 pm, and in some cases the relative order of the values was reversed at this time. Low doses of dietary cholest-4-en-3-one inhibited sterol synthesis and HMG-CoA reductase in livers of all strains tested, whereas high doses or prolonged feeding of the steroid caused a relatively rapid elevation of both sterol synthesis and enzyme activity to above normal levels in several mouse strains including C57L/J. Sterol synthesis and enzyme activity in strain A/HeJ mice were depressed by dietary cholest-4-en-3-one under all conditions tested except when the steroid was fed at a low level for a prolonged period. LAF₁ offspring of the cross C57L/J×A/HeJ responded to dietary cholest-4-en-3-one as did the A/HeJ parental strain. Analysis of the effects of cholest-4-en-3-one upon sterol synthesis in backcross offspring of the mating LAF₁/J×C57L/J did not permit precise estimation of the number of genes that determine the difference in response to the dietary steroid but did suggest that the number may be relatively small.This paper was presented at a symposium entitled Genetic Control of Mammalian Metabolism held at The Jackson Laboratory, Bar Harbor, Maine, June 30–July 2, 1969. The symposium was supported in part by an allocation from NIH General Research Support Grant FR 05545 from the Division of Research Resources to The Jackson Laboratory.Supported in part by NIH Research Grant CA 02758 and NIH Training Grant Tol CA 05013, both from the National Cancer Institute. Some of this work was presented by R.M.P. in partial fulfillment of the requirement for a M.S. degree.     

Evidence for a constitutive and inducible form of kynurenine formamidase in an actinomycin-producing strain of Streptomyces parvulus

Two forms of kynurenine formamidase have been found in an actinomycin-producing strain of Streptomyces parvulus. Formamidase I has a molecular weight of 42,000 and is synthesized constitutively. Formamidase II is smaller (24,000) and is present just prior to and during synthesis of actinomycin.     

Genetic control of glucokinase activity in mice

Inbred strains of mice were surveyed for liver glucokinase activity. Mice of all strains studied could be distributed into three groups with high, intermediate, and low levels of enzyme activity. Genetic analysis using crosses and backcrosses with prototype high (C3H/HeJ) and low (RF/J) strains revealed that glucokinase activity was controlled by a single gene. The name glucokinase and gene symbol Gk are suggested for this gene. The Gk ^a allele designates the strain with high glucokinase activity, while Gk ^b represents the allele in the strain with the low enzyme activity. The interaction of fasting and diabetes on the activity of glucokinase in these two strains is described.Supported in part by United States Public Health Service Research Grant CA 05873 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster

The molecular weight forms of kynurenine formamidase were studied both genetically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH₄)₂SO₄ and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its K _m is 1.87×10^–3 m. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its K _m is 2.31×10^–3 m. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A–27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B–93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.This work was supported by USPHS Grant GM-21286.     

Purification and characterization of two allelic forms of l-glycerol 3-phosphate dehydrogenase from inbred strains of mice

l-Glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) was purified from the muscle of BALB/cJ and C57BL/6J mice. The half-lives of the enzyme at 50 C were 6 and 33 min, respectively, for the BALB/cJ and C57BL/6J strains. Enzyme preparations from the two strains of mice were compared with respect to the following properties and found to be essentially indistinguishable: K _m values for dihydroxyacetone phosphate, NADH, l--glycerophosphate, and NAD⁺; maximum velocity; competitive inhibition by inorganic phosphate; pH optimum; energy of activation; electrophoretic mobility; molecular weight and subunit molecular weight. From these data, it is concluded that the kinetic properties of the purified enzyme are not the factors responsible for the differences in activity found in crude homogenates of mouse tissues.This work was supported by NIH Research Grant HD 06712 from the National Institute of Child Health and Human Development and by an allocation from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Identification of inbred strains of mice,Mus musculus. I. Genetic control of inbred strains of mice using starch gel electrophoresis

Fifteen biochemical markers were tested in 30 inbred strains of mice to control the genetic constitution of each strain. Discrepancies in pattern from Standardized Nomenclature for Inbred Strains of Mice are reported and discussed.This work was supported by Grant No. 512–2532 from the Danish Medical Research Council.     

The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.     

Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14

A single formamidase, which is different from the formamidases found in other tissues, occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity: For-5 ^b is relatively heat stable and is present in strain C57BL/6J, while For-5 ^d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studied to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5 ^d allele is associated with the Es-10 ^b allele, and that the For-5 ^b allele is associated with Es-10 ^a and Es-10 ^c. Formamidase-5 segregates as expected in the F₂ generation of crosses between strains bearing For-5 ^b and For-5 ^d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.This research was sponsored in part by the Department of Energy under contract with the Union Carbide Corporation and in part by NIH Research Grant GM-18684 from the National Institute of General Medical Sciences. J. C. F. is a predoctoral Fellow supported by Grant CA 09104 from the National Cancer Institute. The Biology Division of Oak Ridge National Laboratory and the Jackson Laboratory are fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The Baseline Cosinus Function: A Periodic Regression Model for Biological Rhythms

Abstract

Both tryptophan oxygenase and tyrosine aminotransferase of rat liver show diurnal variations in the inducibility by quinolinic acid. The maxima of effectiveness of quinolinic acid precede the maxima of normal enzyme activity. In the case of tyrosine aminotransferase, the induction kinetics and the dose response curve were also greatly depending on the time of day. No rhythmicity could be detected in the activities of 3‐hydroxyanthranilate oxygenase and ornithine aminotransferase.     

Erythrocyte phosphofructokinase in rat strains with genetically determined differences in 2,3-diphosphoglycerate levels

We have studied the erythrocyte enzyme phosphofructokinase (PFK) from two strains of Long-Evans rats with genetically determined differences in erythrocyte 2,3-diphosphoglycerate (DPG) levels. The DPG difference is due to two alleles at one locus. With one probable exception, the genotype at this locus is always associated with the hemoglobin (Hb) electrophoretic phenotype, due to a polymorphism at the ^III-globin locus. The enzyme PFK has been implicated in the DPG difference because glycolytic intermediate levels suggest that this enzyme has a higher in vivo activity in High-DPG strain rats, although the total PFK activity does not differ. We report here that partially purified erythrocyte PFK from Low-DPG strain cells is inhibited significantly more at physiological levels of DPG (P<0.01) than PFK from High-DPG strain erythrocytes. Citrate and adenosine triphosphate also inhibit the Low-DPG enzyme more than the High-DPG enzyme. Therefore, a structurally different PFK, with a greater sensitivity to inhibitors, may explain the lower DPG and ATP levels observed in Low-DPG strain animals. These data support a two-locus (Hb and PFK) hypothesis and provide a gene marker to study the underlying genetic and physiologic relationships of these loci.This investigation was supported in part by Grant AM 14898, National Research Service Award 5 F 32 AM 05418, and Biochemical Research Support Grant 5 S07 RR 05551 from the National Institutes of Health.     

Heme metabolism in promastigotes of Leishmania donovani

Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (d-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, -aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax ad pH values of heme oxygenase of promastigotes were found to be 1666 M hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.     

Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/β hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.