Guanosine Formation from Guanine by Bacillus licheniformis

Adenine-auxotrophic mutant of Bacillus licheniformis formed considerable amount of guanosine from guanine. The guanosine formation was stimulated by the addition of penicillin to the growing cells and by the presence of uridine in the crude extract. The crude extract preserved for long time showed the changes of the enzyme actions for added guanine.     

Bacterial Ammeline Metabolism via Guanine Deaminase

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.Ammeline is an intermediate in the bacterial metabolism of melamine (Fig. ​(Fig.1).1). Melamine has become internationally recognized as a chemical adulterant in pet foods and infant formula that caused morbidity and mortality in pets and children (12). In pets, where more than 1,000 deaths have been attributed to melamine poisoning, the composition of the causal kidney precipitate was found to be a 1:1 complex of melamine-cyanuric acid (2, 25). In human babies, melamine-uric acid cocrystals have been identified (11). Feeding animals a mixture of melamine and cyanuric acid or a mixture of melamine, ammeline, ammelide, and cyanuric acid was found to produce acute kidney disease (5). Melamine and cyanuric acid are known to form a highly insoluble, hydrogen-bonded network (33) that can precipitate in the kidneys, causing kidney failure. Since bacterial metabolism of melamine generates cyanuric acid (6, 7, 14), it is possible that bacterial melamine metabolism could contribute to melamine toxicity in some cases.Open in a separate windowFIG. 1.The known metabolic pathway in bacteria for transforming melamine to cyanuric acid.Bacteria metabolize melamine by sequential deamination (4, 6, 7, 14, 30) to ammeline, ammelide, and cyanuric acid (Fig. ​(Fig.1).1). The genes and enzymes involved in the deamination of melamine and ammelide are known. Melamine deaminases (TriA and TrzA) have been purified and characterized (20, 28). The enzymes AtzC (34) and TrzC (7) were shown to be capable of ammelide deamination. Although ammeline deamination has been observed in a large number of microbial strains (37), the genes and enzymes involved in bacterial ammeline deamination have remained obscure. Many of the bacteria and fungi that were shown to deaminate ammeline did not deaminate melamine or ammelide (37), indicating that these ammeline deaminating enzymes have not evolved as a component of the melamine degradation pathway.Enzymes functioning in the metabolism of the s-triazine herbicide atrazine are related to some of the enzymes in the melamine pathway. TriA (melamine deaminase) is related to AtzA (28, 31) and TrzN (35), enzymes that catalyze the dechlorination of atrazine. AtzB, which catalyzes the second step in the atrazine metabolic pathway, was reported to also deaminate ammeline as a side reaction, but the rate of the reaction was not measured in that study (27).The enzymes involved in melamine and ammelide deamination, along with other enzymes acting on s-triazine herbicides, are all members of the amidohydrolase superfamily (32, 36). These enzymes typically contain one or two metal ions that are involved in activating water for nucleophilic displacement reactions. A significant number of amidohydrolase superfamily members catalyze deamination reactions with nitrogen heterocyclic ring substrates. In this context, we specifically analyzed the amidohydrolase enzymes in ammeline-metabolizing bacteria to identify the enzyme responsible for the activity. Molecular genetic, biochemical, and in silico data support the hypothesis that guanine deaminase functions as the principal ammeline deaminase activity of bacteria. This has implications for enzyme catalytic promiscuity and understanding the bacterial metabolism of melamine. The latter may be relevant to melamine toxicity in humans and animals.     

Distribution of Guanine Deaminase in Mouse Brain

Guanine deaminase was measured in nearly 100 different areas of mouse brain. The levels are relatively high in all parts of the telencephalon, both gray and white. It is especially active in parts of the olfactory tubercle and amygdala. Levels in the diencephalon range from low to as high as in the telencephalon. Brain areas caudal to the diencephalon, including all parts of the cerebellum, are almost uniformly below the level of detection. The enzyme is also virtually absent from the retina. The extreme range of concentration suggests that guanine deaminase might play a role in the metabolism of a neuroeffector.     

One-Step Protection of the Guanine Moiety in 2′-Deoxyguanosine and Guanosine

Abstract

2′-Deoxyguanosine reacts with 4-nitrophenylsulphonylethene to give a protected nucleoside derivative. Deprotection can be achieved by treatment with concentrated aqueous ammonia. The applicability of the protective group is shown by the synthesis of dT₄G.     

Guanine Deaminase Functions as Dihydropterin Deaminase in the Biosynthesis of Aurodrosopterin, a Minor Red Eye Pigment of Drosophila

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 (k_cat/K_m) for guanine (8.6 × 10⁶ m⁻¹·s⁻¹) was 860-fold higher than that for 7,8-dihydropterin (1.0 × 10⁴ m⁻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) and subsequently by Viscontini et al. (2), consist of at least five compounds, which have been referred to as drosopterin, isodrosopterin, neodrosopterin, aurodrosopterin, and “fraction e” (3). 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). 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), 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).Open in a separate windowSCHEME 1.Proposed pathway for the biosynthesis of drosopterins in D. melanogaster. The red eye pigments in Drosophila are collectively called drosopterins. Drosopterin (a major red eye pigment; labeled D) is produced nonenzymatically by the one-to-one condensation of 7,8-dihydropterin and PDA (10). Aurodrosopterin (a minor red eye pigment; labeled A), which has one less amino group in the pteridine portion of the structure, was shown to be produced nonenzymatically by the condensation of 7,8-dihydrolumazine and PDA (5).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). 7,8-Dihydroneopterin triphosphate is then converted to 6-pyruvoyl tetrahydropterin (6-PTP)³ by PTP synthase, the product of the purple gene (7–9). 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, 11). Aurodrosopterin and its enantiomer, isoaurodrosopterin, are produced nonenzymatically by the one-to-one condensation of 7,8-dihydrolumazine and PDA under acidic conditions (5) 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).In the course of investigating the metabolic fate of tetrahydrobiopterin, Rembold and co-workers (12) 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) 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.     

Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf. We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages. Here, we describe the camouflage1 （cfl） mutant, which develops nonclonal, yellow-green sectors in its leaves. We cloned the cfl gene by transposon tagging and determined that it encodes porphobilinogen deaminase （PBGD）, an enzyme that functions early in chlorophyll and heme biosynthesis. While PBGD has been characterized biochemically, no viable mutations in this gene have been reported in plants. To investigate the in vivo function of PBGD, we characterized the cfl mutant. Histological analyses revealed that cfl yellow sectors display the novel phenotype of bundle sheath cell-specific death. Light-shift experiments determined that constant light suppressed cfl sector formation, a dark/light transition is required to induce yellow sectors, and that sectors form only during a limited time of leaf development. Biochemical experiments determined that of 1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Furthermore, the cfl yellow regions displayed a reduction in catalase activity. A threshold model is hypothesized to explain the cfl variegation and incorporates photosynthetic cell differentiation, reactive oxygen species scavenging, and PBGD function.     

Purification and Properties of Adenosine Nucleosidases from Tea Leaves

Three adenosine nucleosidases (adenosine ribohydrolase, EC 3.2.2.7) with high substrate specificity were isolated from the extracts of tea leaves by a procedure including fractionation with ammonium sulfate, column chromatography on DEAE- and CM-cellulose, and gel filtration on Sephadex G-100. They were designated adenosine nucleosidase I, II and III, respectively, and their properties were characterized.

Among the naturally occurring nucleosides only adenosine and 2′-deoxyadenosine were hydrolyzed by these three enzymes and cleavage rate of the N-glycosidic bond in 2′-deoxyadenosine was three or four times greater than that in adenosine.     

Antioxidants from Steamed Used Tea Leaves and Their Reaction Behavior

The most efficient steaming conditions below 200 °C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (?)-catechin, (?)-epicatechin, (?)-gallocatechin, (?)-epigallocatechin, (?)-catechin gallate, (?)-epicatechin gallate, (?)-gallocatechin gallate, (?)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants.     

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis

Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed.     

茶树叶绿体及其蛋白的分离研究

茶树叶片叶绿体的有效分离纯化是进行茶树叶绿体代谢组学和蛋白质组学研究的基础.本文以茶树鲜叶为材料,通过叶绿体得率、希尔反应等纯度和完整度指标,比较了Percoll密度梯度离心法和蔗糖密度梯度离心法对叶绿体分离纯化的效果;通过蛋白质含量和SDS-PAGE电泳图谱,比较了涨破法和冻融法对叶绿体蛋白的提取效果.结果发现Per...     

The Formation of Aldehydes from Amino Acids by Tea Leaves Extracts

In the reaction system containing amino acid, tea leaves extract and (?)-epicatechin, some amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine and phenylalanine produced formaldehyde, acetaldehyde, isobutyraldehyde, isovaleraldehyde, 2-methylbutanal, methional and phenylacetaldehyde, respectively. The production of these aldehydes was regarded to proceed as Strecker degradation. On the production of phenylacetaldehyde it was revealed in the tea leaves extract-phenol-phenylalanine system that: 1) di-phenol was the most effective co-factor in comparison with mono- and tri-phenols; 2) the optimum concentration of (?)-epicatechin was 5×10-⁴M and the production was depressed at the concentration more than 5×l0-⁴M; 3) the production decreased by diluting tea leaves extract.     

Removal of Heavy Metals by Waste Tea Leaves from Aqueous Solution

In this paper, tea leaves were shown to be an effective, low‐cost biosorbent. Removal of lead, iron, zinc and nickel from 20 mg/L metal solution by dried biomass of waste tea leaves amounted to 96, 91, 72 and 58 %, respectively, at equilibrium, which followed Langmuir and Freundlich adsorption isotherms. Adsorption of metal was in the order of Pb > Fe > Zn > Ni from 5–100 mg/L of metal solution. From a multi‐metallic mixture, 92.5, 84 and 73.2 % of lead, iron and zinc, respectively, were removed. Fourier transform infrared (FTIR) studies indicated that the carboxyl group was involved in the binding of lead and iron, whereas the amine group was involved in the binding of nickel and zinc. A flow through sorption column packed with dried biomass demonstrated a sorption capacity of 73 mg Pb/g of biomass, indicating its potential in cleaning metal containing wastewater. The metal laden biomass obtained could be disposed off by incineration.     

The Epidermal Cells of Tea Leaves

The upper epidrmis of tea leaf consists of cells about 30 40 μ in diameter, with slightly sinuous cell surface and devoid of stomata or hairs. The lower epidermis consists of cells about 50–70 μ in diameter, with more sinuous walls. Stomata confined to the lower surface, surrounded by 3–4 round, subsidiary cells. The upper andlower epidermis of the wild tea of southwest China show the difference in surface texture. Luxuriant wax of the epidermis is in knob or club shape. There are two types of stomata (namely general stomata and stomata of sunken crypts (gland scale)) on the same leaf. The numbers of stomata are distributed 70–100/mm2. Hairs are short and rare, or none. Intercellular flanges between epidermis is steep and thick in wild tea. The protruded parts of the torus are in the form of “foot”. The flanges of the cuticle are rather deep.     

Changes in Volatile C6-Aldehydes Emitted from and Accumulated in Tea Leaves

C₆-Aldehydes emitted from intact tea leaves were analyzed quantitatively.Emission of the aldehydes increased temporarily in mid-May whenenzymatic activities involved in aldehyde formation from lipidsbegan to increase. Levels of C₆-aldehydes in tea leaves alsoincreased temporarily. However, the accumulated C₆-aldehydesdid not always correspond to emitted ones. (Received December 1, 1991; Accepted March 18, 1992)     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Studies on the Flavonoids in Tea Leaves

Yellow needle crystals, C₂₁H₂₀O₁₃?H₂O have been isolated from tea leaves. The crystals yield myricetin (hexaacetate, m.p. 211~212°C), glucose and galactose on hydrolysis. As analytical data indicate the molecular ratio of myricetin to the sugars to be 1:1 and the only bonding position of the sugars to be position 3 of the aglycone, the crystals are concluded to consist of two kinds of glycosides, namely myricetin-3-glucoside and myricetin-3-galactoside.     

An Enzyme Hydrolyzing l-Theanine in Tea Leaves

Theanine hydrolase activity in tea leaves was assayed by measuring enzymatically released ethylamine from l-theanine. The o-phthalaldehyde derivative of ethylamine was measured by reverse phase HPLC recorded with a spectrofluorometric detector.

Theanine hydrolase activity was purified about 4.6-fold by DEAE-cellulose column chromatography. Although this active fraction also had glutaminase activity, the yield of the glutaminase activity was about 50% of that of theanine hydrolytic activity. The theanine hydrolytic activity was inhibited by acidic amino acid and l-alanine, and stimulated by l-malic acid. The purified enzyme solution hydrolyzed not only theanine but also γ-glutamylmethylamide, γ-glutamyl-n-propylamide, γ-glutamyl-n-butylamide, γ-glutamyl-iso-butylamide, and γ-glutamyl-n-amylamide, which were synthesized from l-pyroglutamic acid and corresponding alkylamines. However, N-methylpropionamide and N-ethylpropionamide were not hydrolyzed. The theanine hydrolase activity and glutaminase in tea leaves showed the same pH optimum (8.5).

The activity of theanine hydrolase in tea leaves increased during the first lOhr after plucking but then decreased gradually, while that of glutaminase decreased constantly and was almost lost