FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061530497> ?p ?o ?g. }

• W2061530497 endingPage "23435" @default.
• W2061530497 startingPage "23426" @default.
• W2061530497 abstract "Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 °C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (kcat/Km) for guanine (8.6 × 106m−1·s−1) was 860-fold higher than that for 7,8-dihydropterin (1.0 × 104m−1·s−1). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 °C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (kcat/Km) for guanine (8.6 × 106m−1·s−1) was 860-fold higher than that for 7,8-dihydropterin (1.0 × 104m−1·s−1). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. The complexity of the eye color phenotypes of the fruit fly Drosophila melanogaster has been the subject of numerous investigations for more than 90 years. Two classes of pigments contribute to the eye color of Drosophila: brown “ommochromes” and red “drosopterins.” Drosopterins, first reported by Lederer (1.Lederer E. Biol. Rev. Camb. Philos. Soc. 1940; 15: 273-306Crossref Scopus (16) Google Scholar) and subsequently by Viscontini et al. (2.Viscontini M. Hadorn E. Karrer P. Helv. Chim. Acta. 1957; 40: 579-585Crossref Scopus (26) Google Scholar), consist of at least five compounds, which have been referred to as drosopterin, isodrosopterin, neodrosopterin, aurodrosopterin, and “fraction e” (3.Schwinck I. Genetics. 1971; 68: s59-s60Crossref PubMed Google Scholar). Among the red pigments, drosopterin and isodrosopterin are the major components, whereas aurodrosopterin and neodrosopterin are minor pigments in wild type flies. The chemical structure of drosopterin was determined by Pfleiderer and co-worker (4.Theobald N. Pfleiderer W. Chem. Ber. 1978; 111: 3385-3402Crossref Scopus (25) Google Scholar). Drosopterin and its enantiomer, isodrosopterin, consist of a pentacyclic ring system containing a 5,6,7,8-tetrahydropterin (=2-amino-5,6,7,8-tetrahydropteridin-4(1H)-one), a 2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b][1,4]diazepin-4-one, and a pyrrole ring (Scheme 1). Based on 1H NMR and UV/visible spectral analyses, the structure of aurodrosopterin was elucidated in 1993 by Yim et al. (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar), who found that it is the same as that of drosopterin except that it has one less amino group in the pteridine portion. The presence or absence of an amino group in the pteridine moiety is the key characteristic that distinguishes drosopterin from aurodrosopterin (Scheme 1). They also reported the presence of isoaurodrosopterin based on thin layer chromatographic analyses of Drosophila head extracts using various solvent systems (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar). The first step leading to the biosynthesis of drosopterins is the formation of 7,8-dihydroneopterin triphosphate from GTP by GTP cyclohydrolase I, which is encoded by Punch (6.Mackay W.J. O'Donnell J.M. Genetics. 1983; 105: 35-53Crossref PubMed Google Scholar). 7,8-Dihydroneopterin triphosphate is then converted to 6-pyruvoyl tetrahydropterin (6-PTP) 3The abbreviations used are: 6-PTP6-pyruvoyl tetrahydropterinpterin2- amino-4-hydroxypteridinePDApyrimidodiazepineHPLChigh performance liquid chromatographyTLCthin layer chromatographyMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flight.3The abbreviations used are: 6-PTP6-pyruvoyl tetrahydropterinpterin2- amino-4-hydroxypteridinePDApyrimidodiazepineHPLChigh performance liquid chromatographyTLCthin layer chromatographyMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flight. by PTP synthase, the product of the purple gene (7.Yim J.J. Grell E.H. Jacobson K.B. Science. 1977; 198: 1168-1170Crossref PubMed Scopus (30) Google Scholar, 8.Krivi G.G. Brown G.M. Biochem. Genet. 1979; 17: 371-390Crossref PubMed Scopus (35) Google Scholar, 9.Park Y.S. Kim J.H. Jacobson K.B. Yim J.J. Biochim. Biophys. Acta. 1990; 1038: 186-194Crossref PubMed Scopus (18) Google Scholar). Next 6-PTP is converted to pyrimidodiazepine (PDA) by PDA synthase, which is a member of the Omega class glutathione S-transferases and is encoded by the sepia gene (10.Wiederrecht G.J. Paton D.R. Brown G.M. J. Biol. Chem. 1984; 259: 2195-2200Abstract Full Text PDF PubMed Google Scholar, 11.Kim J. Suh H. Kim S. Kim K. Ahn C. Yim J. Biochem. J. 2006; 398: 451-460Crossref PubMed Scopus (44) Google Scholar). Aurodrosopterin and its enantiomer, isoaurodrosopterin, are produced nonenzymatically by the one-to-one condensation of 7,8-dihydrolumazine and PDA under acidic conditions (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar) in a manner similar to the production of drosopterin and isodrosopterin, which are produced by a similar nonenzymatic condensation of 7,8-dihydropterin and PDA (Scheme 1). 6-pyruvoyl tetrahydropterin 2- amino-4-hydroxypteridine pyrimidodiazepine high performance liquid chromatography thin layer chromatography matrix-assisted laser desorption/ionization time-of-flight. 6-pyruvoyl tetrahydropterin 2- amino-4-hydroxypteridine pyrimidodiazepine high performance liquid chromatography thin layer chromatography matrix-assisted laser desorption/ionization time-of-flight. In the course of investigating the metabolic fate of tetrahydrobiopterin, Rembold and co-workers (12.Rembold H. Metzger H. Gutensohn W. Biochim. Biophys. Acta. 1971; 230: 117-126Crossref PubMed Scopus (28) Google Scholar) found that tetrahydrobiopterin can be degraded to 6-hydroxylumazine by rat liver homogenates. They proposed that tetrahydrobiopterin was converted by nonenzymatic side chain release to 7,8-dihydropterin, which was converted to 7,8-dihydrolumazine, the deaminated counterpart of 7,8-dihydropterin, by a deaminase present in the crude extracts. 7,8-Dihydrolumazine is then converted to 7,8-dihydro-6-hydroxylumazine by xanthine oxidase and subsequently to 6-hydroxylumazine by autoxidation. This series of reactions was also observed in D. melanogaster. Takikawa et al. (13.Takikawa S. Tsusue M. Gyure W.L. Insect Biochem. 1983; 13: 361-368Crossref Scopus (2) Google Scholar) demonstrated the conversion of 7,8-dihydropterin to 6-hydroxylumazine using partially purified fly extracts. However, the enzymatic properties of the deaminase and the identity of the gene encoding the protein have not yet been established. Here we purified and characterized Drosophila dihydropterin deaminase and identified its structural gene, CG18143, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. We provide clear evidence that CG18143, previously annotated as the guanine deaminase gene, is directly involved in the biosynthesis of aurodrosopterin, a minor red eye pigment in Drosophila. The samples of 7,8-dihydrolumazine and PDA were generously gifted to us by Professor Wolfgang Pfleiderer of Konstanz University in Konstanz, Germany. 7,8-Dihydroneopterin triphosphate was prepared enzymatically from GTP by GTP cyclohydrolase I purified from Escherichia coli according to a previously published procedure (14.Yim J.J. Brown G.M. J. Biol. Chem. 1976; 251: 5087-5094Abstract Full Text PDF PubMed Google Scholar). Purified 7,8-dihydropterin was prepared from pterin by zinc-alkali reduction according to previously described methods (15.Kaufman S. J. Biol. Chem. 1967; 242: 3934-3943Abstract Full Text PDF PubMed Google Scholar). Drosopterin, isodrosopterin, aurodrosopterin, and neodrosopterin were isolated from Drosophila heads by methods published earlier (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar). Other pteridines were purchased commercially from Schircks Laboratories (Jona, Switzerland). A wild type of D. melanogaster, Oregon-R, was used for the preparation of the enzymes. The breeding populations of the flies were maintained at 25 ± 1 °C in a culture cage on a standard yeast medium with propionic acid added as a mold inhibitor. For the purification of deaminase, adult flies (0–2 days old) were frozen at −70 °C until used. The fly strains used for the mutational analysis included the following. y1 w67c23;;P{y+mDint2 wBR.E.BR SUPor-P} KG04159 (16.Roseman R.R. Johnson E.A. Rodesch C.K. Bjerke M. Nagoshi R.N. Geyer P.K. Genetics. 1995; 141: 1061-1074Crossref PubMed Google Scholar) was obtained from the Bloomington Stock Center (Department of Biology, Indiana University, Bloomington, IN), and ;;Df(3R)A321R1,ry (81F; 82A4-6) was a gift from S. A. Wasserman (University of California, San Diego, CA). For the 7,8-dihydropterin deaminase assay, the standard reaction mixture contained 0.5 mm 7,8-dihydropterin, 100 mm potassium phosphate buffer (pH 7.5), and the enzyme in a final volume of 50 μl. After incubation at 40 °C for 10–30 min in the dark, the reaction was stopped by the addition of 5 μl of 30% trichloroacetic acid, and the mixture was centrifuged to remove the precipitated proteins. A 6.5-μl iodine solution (1% I2, 2% KI) was then added to the acidified reaction mixture to convert the reduced pteridines to their oxidized forms. After incubation for 1 h at room temperature, the excess iodine was reduced by the addition of 5 μl of 2% ascorbic acid. A portion of this mixture was subjected to HPLC on a reversed-phase C18 column (Inertsil ODS-3, 4.6 × 250 mm; GL Sciences) connected to a photodiode array detector (Waters 996) and a scanning fluorescence detector (Waters 474). The material was eluted isocratically with 85% (v/v) 50 mm ammonium acetate in 15% (v/v) methanol at a flow rate of 1 ml/min. The reaction products, lumazine and 6-hydroxylumazine, were monitored by their fluorescence intensities. For the guanine deaminase assay, the standard reaction mixture contained 0.1 mm guanine, 100 mm potassium phosphate buffer (pH 7.5), and the enzyme in a final volume of 100 μl and was incubated at 40 °C for 10–30 min in the dark. A portion of this mixture was subjected to HPLC with the same equipment as described above. The material was eluted isocratically with 50 mm sodium acetate buffer (pH 6.0) at a flow rate of 1 ml/min, and xanthine was monitored by the absorbance at 270 nm. This assay was carried out spectrophotometrically as described previously (17.Taras M.J. Arnold E.F. Hoak R.D. Rand M.C. Greenberg A.E. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, D. C1981: 350-352Google Scholar) with some modifications. The reaction mixture contained a 0.25 mm concentration of each substrate, 100 mm potassium phosphate buffer (pH 7.5), and the enzyme in a final volume of 100 μl. After incubation at 40 °C for 30 min in the dark, the reaction was stopped by the addition of 350 μl of cold deionized water, and 2.5 μl of 3 mm MnSO4 solution was added to the sample. After 25 μl of hypochlorous acid reagent (10 ml of 5% NaOCl solution and 40 ml of water, pH 6.8) was added to the mixture and stirred, 30 μl of phenate reagent (2.5 g of NaOH and 10 g of phenol in 100 ml of water) was immediately added. After 10 min, the increase in absorption at 630 nm was measured relative to the absorption of the control solution. The same reaction mixture without the added enzyme was used as the control. Dihydropterin deaminase was purified from head extracts of D. melanogaster by using conventional column chromatography. All of the operations were carried out at 4 °C. After the flies were frozen with liquid nitrogen, their heads were detached from their bodies by mechanical shock and separated from the other parts by sieving. The fly heads (dry weight of 20 g) were homogenized in 80 ml of 0.1 m potassium phosphate buffer (pH 7.0) containing 2 mm phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 25,000 × g for 1 h, and the resulting supernatant was used for the subsequent steps. The proteins precipitated in 30–60% saturated ammonium sulfate were redissolved in 12 ml of 50 mm potassium phosphate buffer (pH 7.0). The solution was then dialyzed twice against 2 liters of the same buffer for 6 h. The dialyzed ammonium sulfate fraction was introduced into a Sephacryl HR S 300 column (2.5 × 78 cm), and the column was eluted with 50 mm potassium phosphate buffer (pH 7.0) at a flow rate of 30 ml/h. The fractions containing the active enzyme were combined (27.5 ml), and solid ammonium sulfate was added to yield a final concentration of 0.1 m. The sample was applied to a phenyl-Sepharose 6 fast flow column (1.5 × 11 cm) that had been equilibrated with 50 mm potassium phosphate (pH 6.1) containing 0.1 m ammonium sulfate. After the column was washed with the same buffer, elution was carried out with a linear gradient of ethylene glycol from 0 to 50% in 25 mm histidine HCl buffer (pH 6.1) at a flow rate of 30 ml/h. The active fractions were combined and concentrated to 5 ml with solid polyethylene glycol (molecular weight = 20,000). This material was dialyzed twice against 1 liter of 25 mm histidine HCl buffer (pH 6.1) for 6 h and loaded on a column of Polybuffer Exchanger 94 (1 × 37 cm) that had been equilibrated previously with 25 mm histidine HCl buffer (pH 6.1). The column was developed with 250 ml of elution buffer (10-fold diluted Polybuffer Exchanger 74, pH 5.0) at a flow rate of 15 ml/h. The active fractions were desalted with 20 mm phosphate buffer (pH 7.0) and concentrated using Amicon Centriplus Centrifugal Filter Devices (Millipore; molecular mass cutoff, 10,000 Da). The condensation reaction was performed as described previously (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar) with some modifications. 7,8-Dihydropterin was incubated with the purified deaminase, and aliquots of the reaction mixture were taken out at various time points and acidified to pH 2.5 by the addition of 2 m HCl. Each acidified mixture was then incubated with PDA, which had been preincubated at pH 2.5 for 30 min, and the synthesized pigments were analyzed by thin layer chromatography (TLC). The initial velocities of the catalytic reactions for 7,8-dihydropterin and guanine were determined under standard reaction conditions at various substrate concentrations. All of the experiments were performed three times. Ten different substrate concentrations were prepared within a range of 25–3000 μm for 7,8-dihydropterin and 10–300 μm for guanine. The initial velocities were measured within a 15–20% consumption range for each substrate. The purified enzyme was subjected to SDS-PAGE, and the gel was silver-stained by a previously described method (18.Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (836) Google Scholar). The protein band was excised and in-gel digested with trypsin according to previously published procedures (19.Arnott D. O'Connell K.L. King K.L. Stults J.T. Anal. Biochem. 1998; 258: 1-18Crossref PubMed Scopus (124) Google Scholar). MALDI-TOF mass spectra were acquired on a Voyager-DETM STR Biospectrometry Workstation. The peptide masses were measured as monoisotopic masses and analyzed against the NCBInr data base using the MS-Fit search algorithm (Protein Prospector, University of California San Francisco Mass Spectrometry Facility, San Francisco, CA). For the data base searches, no restrictions were placed on the species or organisms. Peptides were selected in the mass range of 1000–3000 Da. The coding sequence of CG18143 was amplified by reverse transcription-PCR. The total RNA was extracted from 0–2-day-old adult flies with TRIzol® reagent in accordance with the manufacturer's instructions (Invitrogen). PCR was carried out under the following conditions: one cycle of 94 °C for 4 min; 30 cycles of 94 °C for 1 min, 48 °C for 1 min, and 72 °C for 2 min; and one cycle of 72 °C for 7 min. For PCR, we used a gene-specific primer set, DPD1 (5′-GGGCCTTCCATATGGCAACCGTGTTTCTTGGAACT-3′) and DPD2 (5′-CGCGGATCCTCATTGGTATCCCTGTTTAATACGC-3′), designed to incorporate the NdeI and BamHI sites (underlined), respectively. The PCR products were subcloned into the multiple cloning site of the pET 15b vectors and transformed into BL21 cells. Transformants were cultured at 37 °C in 1000 ml of LB medium containing 50 μg/ml ampicillin, and protein expression was induced by the addition of 0.5 mm isopropyl d-thiogalactopyranoside at OD 0.5. After 3 h, the cells were collected by centrifugation at 5000 × g for 10 min and suspended in 40 ml of ice-cold Binding Buffer (pET system manual, Novagen). After sonication and centrifugation, the supernatant containing the soluble enzyme was bound to His·Bind Resin according to the manufacturer's protocol (Novagen). For Southern blot analysis, genomic DNA extracted from ;;P{y+mDint2 wBR.E.BR SUPor-P} flies was cut by EcoRI or by a combination of EcoRI and EcoRV, separated on 1% agarose gels, and blotted onto Nytran® N membranes (Schleicher & Schuell). The blotted membranes were probed with a 32P-labeled DNA probe covering the 5′ end of the SUPor-P element using QuikHyb® Hybridization Solution (Stratagene) and autoradiographed. The inverse PCR method 4E. J. Rehm, unpublished. was used to obtain the genomic sequence flanking the insertion site of the P element in the ;;P{y+mDint2 wBR.E.BR SUPor-P} flies. A Plac1 (5′-CACCCAAGGCTCTGCTCCCACAAT-3′) and Plac4 (5′-ACTGTGCGTTAGGTCCTGTTCATTGTT-3′) primer set was used for the 5′ junction, and a Pry1 (5′-CCTTAGCATGTCCGTGGGGTTTGAAT-3′) and Pry4-added (5′-CAATCATATCGCTGTCTCACTCAGACT-3′) primer set was used for the 3′ junction. The amplified DNA fragments were directly sequenced using the Plac1 and Pry1 primers. The sequences were analyzed with basic local alignment search tool (BLAST) searches of the Drosophila Genome Database. After the insertion site of the SUPor-P element was identified, genomic DNA isolated from ;;SUPor-P/Df(3R)A321R1 flies was analyzed by PCR to determine whether the deficient region of ;;Df(3R)A321R1,ry (81F; 82A4-6) flies contains CG18143. A pair of primers that covers the CG18143 and P element insert region was used: Df-5′-primer (5′-TCGTCCACTGCCAGGAATCCTCCTTC-3′) and Df-3′-primer (5′-TATGTACAGGTGCGAGCAGTGCA-3′). The PCR conditions were as follows: one cycle of 94 °C for 4 min; 30 cycles of 94 °C for 1 min, 57 °C for 50 s, and 72 °C for 2 min; and one cycle of 72 °C for 7 min. A male of y1 w67c23 ;;P{y+mDint2 wBR.E.BR SUPor-P} was crossed to ;;TM3/TM6b females. One ;;SUPor-P/TM3 male resulting from this cross was crossed again to ;;TM3/TM6b females. From the cross between the males and ;;SUPor-P/TM3 females of the F2 generation, ;;SUPor-P homozygotes without the y1 mutation in the X chromosome were obtained. To delete the ry mutation from Df(3R)A321R1,ry flies, a recombination method was applied. First ;;Df(3R)A321R1,ry/TM3 males were crossed to Oregon-R virgins, and ;;Df(3R)A321R1,ry/+ virgins were then crossed to ;;TM3/TM6b males. Single male progeny carrying a TM6b balancer were mated in individual vials to ;;Df(3R)A321R1,ry/TM3 females to determine whether the males had a deficient third chromosome. Stocks were established by mating males and females carrying a TM3 balancer. Finally the males carrying a deficient third chromosome were crossed to ry506 females to determine whether the ry mutation on the third chromosome of the deficient fly was deleted. To get ;;Df(3R)A321R1/+, ;;SUPor-P/+, and ;;SUPor-P/Df(3R)A321R1 flies, Df(3R)A321R1/TM3 males were crossed with Oregon-R virgins, and ;;SUPor-P homozygote males were crossed with Oregon-R and ;;Df(3R)A321R1/TM3 virgins. The xanthine and uric acid contents were determined by HPLC using a previously described method (20.Hilliker A.J. Duyf B. Evans D. Phillips J.P. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 4343-4347Crossref PubMed Scopus (112) Google Scholar). Separations were performed on a 4.6 × 250-mm Inertsil ODS-3 reversed-phase column. Uracil, xanthine, and uric acid were monitored at 260, 273, and 292 nm, respectively. The protein concentration was determined by the Bradford method (21.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) with bovine serum albumin as the standard protein. The molecular mass of the native enzyme was estimated using a Superose S 12 column (1.0 × 40 cm) calibrated with standard molecular mass proteins and blue dextran (2,000,000 Da). SDS-PAGE was performed on 4–20% SDS-polyacrylamide gradient gels, and isoelectric focusing was carried out as described previously (22.Awdeh Z.L. Williamson A.R. Askonas B.A. Nature. 1968; 219: 66-67Crossref PubMed Scopus (400) Google Scholar) on a polyacrylamide flat bed gel (0.1 × 14 × 15 cm). TLC was performed with microcrystalline cellulose plates (Eastman Kodak Co. Number 13255 without a fluorescence indicator). For one-dimensional TLC, a 3% ammonium chloride solution was used as a solvent. For two-dimensional TLC, isopropanol, 2% ammonium acetate (1:1, v/v) and 3% ammonium chloride solutions were used as the first and second developing solvents, respectively. Fluorescent spots were detected by a long wave UV lamp. Dihydropterin deaminase from the heads of Drosophila was purified 5850-fold to apparent homogeneity. A summary of this purification is presented in Table 1. Throughout all of the column chromatographic steps, a single activity peak was observed, and the degree of purification was monitored by the increase in the specific activity and by SDS-PAGE (Fig. 1A). The enzyme was purified to a specific activity of 643 units/mg with an activity yield of 4.5%; more than 30 μg of pure protein was routinely obtained from 20 g (dry weight) of 0–2-day-old adult heads. The final preparation of the enzyme did not contain xanthine oxidase/dehydrogenase activity, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydro-6-hydroxypterin. The molecular mass of the purified enzyme was estimated to be 48 kDa by 4–20% gradient SDS-PAGE (Fig. 1A) and by size exclusion chromatography on a calibrated Superose S 12 HPLC column (Fig. 1B), indicating that it is a monomer under native conditions.TABLE 1Summary of purification of dihydropterin deaminaseEnzyme preparationTotal proteinSpecific activityActivity yieldRelative specific activitymgunits/mg%Crude extract46170.111001.0Ammonium sulfate fraction25000.1681.31.5Sephacryl HR S 300 eluate2111.0845.99.8Phenyl-Sepharose 6 Fast Flow eluate9.518.435.2167Polybuffer Exchanger 94 eluate0.032643.54.55850 Open table in a new tab Forrest et al. (23.Forrest H.S. van Baalen C. Viscontini M. Piraux M. Helv. Chim. Acta. 1960; 43: 1005-1010Crossref Scopus (7) Google Scholar) showed that the 5,6-double bonds of 7,8-dihydrolumazine are readily susceptible to nucleophilic attack, e.g. water addition. According to previous reports (12.Rembold H. Metzger H. Gutensohn W. Biochim. Biophys. Acta. 1971; 230: 117-126Crossref PubMed Scopus (28) Google Scholar), 7,8-dihydrolumazine is converted to 7,8-dihydro-6-hydroxylumazine either in the presence or in the absence of xanthine oxidase, and 7,8-dihydro-6-hydroxylumazine is converted to 6-hydroxylumazine by auto-oxidation. We monitored the enzymatic deamination reaction of 7,8-dihydropterin at different time points (zero time, 20 min, and 12 h) by HPLC. After 20 min of the enzymatic reaction, three peaks were detected, the retention times of which were 4.4, 7.0, and 7.5 min, identical to those of standard 6-hydroxylumazine, lumazine, and pterin, respectively. The UV-visible spectra of these peaks were also identical to those of standard 6-hydroxylumazine, lumazine, and pterin, respectively (data not shown). When the enzymatic reaction was prolonged to 12 h, only 6-hydroxylumazine and pterin peaks were detected with the lumazine peak hardly detected (Fig. 2A, lower panel). These results indicate that 7,8-dihydrolumazine is formed from 7,8-dihydropterin by the enzyme and then nonenzymatically hydroxylated to 7,8-dihydro-6-hydroxylumazine, which is converted to 6-hydroxylumazine by iodine oxidation. To identify the deaminase reaction product, we also tried to synthesize aurodrosopterin, which is produced nonenzymatically by the one-to-one condensation of 7,8-dihydrolumazine and PDA under acidic conditions (5.Yim J. Kim S.J. Walcher G. Pfleiderer W. Helv. Chim. Acta. 1993; 76: 1970-1979Crossref Scopus (9) Google Scholar). For this purpose, aliquots of the deaminase reaction mixture taken out at various time points were incubated with PDA as described under “Experimental Procedures.” The synthesized pigments were analyzed by one-dimensional TLC (Fig. 2B). At the early stage of the deamination reaction, only drosopterin and isodrosopterin were detected; as the deamination reaction proceeded, the synthesis of aurodrosopterins gradually increased (Fig. 2B). After the substrate had been fully exhausted, the synthesized pigments were detected at the same position as that of natural aurodrosopterin on a two-dimensional TLC plate (Fig. 2C). Consequently the enzymatic reaction product is 7,8-dihydrolumazine, which is the precursor of aurodrosopterin in nonenzymatic synthesis. Various pteridine compounds were tested for substrate specificity. When the deaminase activity for 7,8-dihydropterin was 100%, the activities for tetrahydropterin, tetrahydrobiopterin, and tetrahydroneopterin were determined to be 71, 62, and 63%, respectively. The enzyme had no deaminase activities for other dihydropteridines such as 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and sepiapterin; it also did not show deaminase activity with fully oxidized pteridines such as pterin, biopterin, neopterin, monapterin, xanthopterin, isoxanthopterin, aminopterin, 6-formylpterin, 6-carboxypterin, and 6-hydroxymethylpterin. Moreover the enzyme did not use folic acid, PDA, drosopterin, or isodrosopterin as a substrate. To determine whether the high reactivities with tetrahydropteridines are due to the nonenzymatic conversion of tetrahydropteridines to 7,8-dihydropterin, the spectral changes in the reaction mixture were examined during the deaminase reaction. When tetrahydropterin was incubated under the standard reaction condition in the absence of enzyme, the UV-visible spectrum of the reaction mixture increased gradually to that of 7,8-dihydropterin up until 20 min. The highest deaminase activity was obtained when the aliquot was taken out at 20 min and used as the source of the substrate. Moreover when the deaminase assay with tetrahydropterin was coupled with 6,7-dihydropteridine reductase (EC 1.5.1.34), which specifically converts quinonoid dihydropteridines to their tetrahydro forms (24.Park D. Park S. Yim J. Biochim. Biophys. Acta. 2000; 1492: 247-251Crossref PubMed Scopus (5) Google Scholar), no deaminase activity was detected. This result strongly suggests that the reason for the high deaminase activities for tetrahydropteridines is that tetrahydropteridines are converted to 7,8-dihydropterin by nonenzymatic side chain release at position 6 via quinonoid dihydropteridines. Interestingly the enzyme exhibited 40-fold higher activities for guanine than for 7,8-dihydropterin (Table 2) along with 60% activity for 8-azaguanine and 5% activity for guanosine relative to 7,8-dihydropterin. No activity was f" @default.
• W2061530497 created "2016-06-24" @default.
• W2061530497 creator A5007023153 @default.
• W2061530497 creator A5026233661 @default.
• W2061530497 creator A5036401519 @default.
• W2061530497 creator A5037165074 @default.
• W2061530497 creator A5054042900 @default.
• W2061530497 date "2009-08-01" @default.
• W2061530497 modified "2023-09-26" @default.
• W2061530497 title "Guanine Deaminase Functions as Dihydropterin Deaminase in the Biosynthesis of Aurodrosopterin, a Minor Red Eye Pigment of Drosophila" @default.
• W2061530497 cites W1539112821 @default.
• W2061530497 cites W1542656360 @default.
• W2061530497 cites W1669877415 @default.
• W2061530497 cites W1875840637 @default.
• W2061530497 cites W1933865442 @default.
• W2061530497 cites W1939943976 @default.
• W2061530497 cites W1972182855 @default.
• W2061530497 cites W1976109604 @default.
• W2061530497 cites W1983925683 @default.
• W2061530497 cites W1992497040 @default.
• W2061530497 cites W2021484484 @default.
• W2061530497 cites W2024957156 @default.
• W2061530497 cites W2026963218 @default.
• W2061530497 cites W2033654734 @default.
• W2061530497 cites W2036969501 @default.
• W2061530497 cites W2039161870 @default.
• W2061530497 cites W2043794222 @default.
• W2061530497 cites W2049230713 @default.
• W2061530497 cites W2053394321 @default.
• W2061530497 cites W2058663113 @default.
• W2061530497 cites W2058986562 @default.
• W2061530497 cites W2066803957 @default.
• W2061530497 cites W2071229329 @default.
• W2061530497 cites W2078322631 @default.
• W2061530497 cites W2082186776 @default.
• W2061530497 cites W2084661072 @default.
• W2061530497 cites W2086070043 @default.
• W2061530497 cites W2089779246 @default.
• W2061530497 cites W2113575788 @default.
• W2061530497 cites W2131306691 @default.
• W2061530497 cites W2137475103 @default.
• W2061530497 cites W2147802260 @default.
• W2061530497 cites W2404898894 @default.
• W2061530497 cites W4293247451 @default.
• W2061530497 doi "https://doi.org/10.1074/jbc.m109.016493" @default.
• W2061530497 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2749116" @default.
• W2061530497 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19567870" @default.
• W2061530497 hasPublicationYear "2009" @default.
• W2061530497 type Work @default.
• W2061530497 sameAs 2061530497 @default.
• W2061530497 citedByCount "11" @default.
• W2061530497 countsByYear W20615304972012 @default.
• W2061530497 countsByYear W20615304972013 @default.
• W2061530497 countsByYear W20615304972014 @default.
• W2061530497 countsByYear W20615304972015 @default.
• W2061530497 countsByYear W20615304972016 @default.
• W2061530497 countsByYear W20615304972017 @default.
• W2061530497 countsByYear W20615304972021 @default.
• W2061530497 crossrefType "journal-article" @default.
• W2061530497 hasAuthorship W2061530497A5007023153 @default.
• W2061530497 hasAuthorship W2061530497A5026233661 @default.
• W2061530497 hasAuthorship W2061530497A5036401519 @default.
• W2061530497 hasAuthorship W2061530497A5037165074 @default.
• W2061530497 hasAuthorship W2061530497A5054042900 @default.
• W2061530497 hasBestOaLocation W20615304971 @default.
• W2061530497 hasConcept C104317684 @default.
• W2061530497 hasConcept C165912504 @default.
• W2061530497 hasConcept C178790620 @default.
• W2061530497 hasConcept C181199279 @default.
• W2061530497 hasConcept C185592680 @default.
• W2061530497 hasConcept C2778301229 @default.
• W2061530497 hasConcept C34628245 @default.
• W2061530497 hasConcept C512185932 @default.
• W2061530497 hasConcept C553450214 @default.
• W2061530497 hasConcept C55493867 @default.
• W2061530497 hasConcept C64584667 @default.
• W2061530497 hasConcept C86803240 @default.
• W2061530497 hasConceptScore W2061530497C104317684 @default.
• W2061530497 hasConceptScore W2061530497C165912504 @default.
• W2061530497 hasConceptScore W2061530497C178790620 @default.
• W2061530497 hasConceptScore W2061530497C181199279 @default.
• W2061530497 hasConceptScore W2061530497C185592680 @default.
• W2061530497 hasConceptScore W2061530497C2778301229 @default.
• W2061530497 hasConceptScore W2061530497C34628245 @default.
• W2061530497 hasConceptScore W2061530497C512185932 @default.
• W2061530497 hasConceptScore W2061530497C553450214 @default.
• W2061530497 hasConceptScore W2061530497C55493867 @default.
• W2061530497 hasConceptScore W2061530497C64584667 @default.
• W2061530497 hasConceptScore W2061530497C86803240 @default.
• W2061530497 hasIssue "35" @default.
• W2061530497 hasLocation W20615304971 @default.
• W2061530497 hasLocation W20615304972 @default.
• W2061530497 hasLocation W20615304973 @default.
• W2061530497 hasLocation W20615304974 @default.
• W2061530497 hasOpenAccess W2061530497 @default.
• W2061530497 hasPrimaryLocation W20615304971 @default.
• W2061530497 hasRelatedWork W1549208733 @default.
• W2061530497 hasRelatedWork W1560471104 @default.

• next