Reaction mixtures contained 200 mM Tris (pH 8.5), 1 mM NADP, 12 μg YdfG, and 0.5 M l-serine or 3-hydroxypropionic acid in a total volume of 400 μl. An ntrB(Con) rutG strain grew slowly on pyrimidine bases (uracil and thymine) and obtained both nitrogens from the ring. Introduction • The biosynthesis of pyrimidine is a simpler process than the purines. Although E. coli does not grow on pyrimidines as the sole nitrogen source at 37°C, it transcribes the rut operon very highly at this temperature under nitrogen-limiting conditions (31, 59). For standard reactions, we used 18 μg of His6-RutA, 6 μg of Fre, and [14C]uracil (radiolabeled at position 2 or 6 at 2 × 107 cpm) in a total volume of 120 μl. Based on our in vitro results, this was expected for strains carrying lesions in rutA but not necessarily for strains carrying rutF lesions because RutF can be replaced in vitro by the flavin reductase Fre. 15N2 We use a variety of approaches other than biochemical assays to explore the functions of RutC, -D, and -E. Although these proteins are not required in vitro, they are required in vivo for growth on uridine as the sole nitrogen source and appear to accelerate removal of toxic intermediates in the Rut pathway or their by-products. The RutB reaction.RutB was initially predicted to be an isochorismatase and later a homologue of N-carbamoylsarcosine amidohydrolase (31, 32). Pyrimidine synthesis is controlled at the first committed step. The 13C and 1H shifts for the RutA/F product were the same as those for synthetic ureidoacrylate (Table 2), and the 1H shifts and J couplings of the synthetic compound agreed well with published values (Table 3). HpaC also has a strong preference for FMN and NADH (19). This superpathway includes the so-called "reductive pathway", in which pyrimidine nucleobases are reduced to a β amino acid, CO 2 and ammonia. 5 and 6). Only half of the Rut enzymes (RutA, RutF, and RutB) are required in vitro to release both nitrogens from the pyrimidine ring as NH4 The requirement for YdfG function or RutE function for utilization of uridine at 37°C was decreased in the UpBCon1 background (i.e., in the presence of the sroG lesion) (Table 7; see Fig. In the latter connection, it will be interesting to determine the identities of rutC and rutD suppressors, for which tools are now available (see Materials and Methods), and to understand why a rutC strain and rutD suppressors go off the pathway and excrete less than the usual amount of 3-hydroxypropionic acid into the medium (see Table S1 in the supplemental material). Work in a related article (37) shows that chemically synthesized ureidoacrylate peracid is rapidly reduced to ureidoacrylate under in vitro reaction conditions similar to ours (20 mM NADH rather than 4 mM and phosphate buffer at pH 8 rather than 7) and presents a plausible mechanism for the formation of ureidoacrylate peracid by RutA. The reductive pathway is found in mammals, plants, some fungi and microorganisms [ Fritzson57, Campbell57, Evans61, Tsai65, Gojkovic00 ]. Cell extracts of UpBCon1 (NCM4384).Cells were grown on minimal medium with glycerol (0.5%) as a carbon source and uridine (5 mM) as the sole nitrogen source at 37°C. (B) Formation and use of ureidoacrylate. The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems † Kwang-Seo Kim,1‡ Jeffrey G. Pelton, 2§‡ William B. Inwood,1 Ulla Andersen, ¶ Sydney Kustu,1* and David E. Wemmer 2 Departments of Plant and Microbial Biology1 and Chemistry,2 University of … Amity Institute of Biotechnology Amity University Rajasthan 2. NMR evidence that RutA cleaves the uracil ring between N-3 and C-4 and incorporates O from O2 at C-4. 3B) (16). Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. The second rutE suppressor (NCM4300), which had the same growth rate on uridine as the first, had a lesion that disrupts the inverted repeat in the binding site for NemR/RutR in the promoter-regulatory region for the nemRA operon. Reaction mixtures were incubated at room temperature with agitation for 20 min, and reactions were stopped by putting them on ice and then freezing them at −20°C. Assay for the RutB protein.The RutB protein was assayed using 14C-labeled RutA product as the substrate and monitoring by TLC as described above or using chemically synthesized ureidoacrylate (16) and monitoring the decrease in absorbance at 266 nm with extinction coefficients of 17,800 M− Cm− in 0.025 M HCl (16) and 18,300 in 40 mM Tris buffer (pH 8.2). Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato ( Solanum tuberosum L.) tubers | SpringerLink The same result was obtained if the RutA, Fre, and RutB proteins were added to radiolabeled uracil simultaneously (Fig. Spectra were taken in DMSO, and data were recorded at 800 MHz. 1). (The YdfG protein, which is coded for outside the rut operon, may not be required under these circumstances.) For samples in panels A and C, reactions were run at pH 8.2, and for samples in panel B, they were run at pH 7. In an ntrB(Con) background (NCM3876), we isolated two strains that could grow on uridine at 37°C, UpBCon1 (NCM4384) and UpBCon2 (NCM4139). 3 A). Wild-type or ntrB(Con) strains with a nonpolar deletion in rutE failed to grow on uridine (Table 5; see Fig. For the ureidoacrylate compounds, an isocratic gradient of 10% acetonitrile, 89% water, and 1% formic acid was used. The extent of the deletion was verified by PCR amplification and sequencing, and the same deletion was found to have occurred independently when a rutE::Kan mutation was introduced into the ntrB(Con) background by phage P1-mediated transduction (Table 1). 5B). In vivo the absence of YdfG results in a growth defect or failure to grow on uridine as the sole nitrogen source in different genetic backgrounds, indicating that E. coli K-12 requires YdfG despite the fact that both moles of ammonium have already been released from the pyrimidine ring before it acts. S4 in the supplemental material) indicates that suppression is likely to be due to increased expression of NemA, as does the finding that NemR apparently controls only nemRA transcription, despite the fact that E. coli contains very large amounts of it (51). In the Pyd pathway, uracil is ﬁrst reduced to dihydrouracil by The different chemical shifts of the species in panels A and B result from the fact that spectrum A was recorded in H2O, whereas spectrum B was recorded in DMSO. Source of Atoms in Pyrimidine Ring; Steps of Pyrimidine Synthesis. Carbamate and aminoacrylate, which hydrolyze spontaneously, are the presumed intermediates. Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. Degradation of 14C-labeled uracil or thymine by cell extracts.To initiate studies of Rut enzymes in vitro, we first prepared cell extracts of NCM4384 (UpBCon1) grown on uridine at 37°C. Synthesis of Z-3-ureido-2-propenoic acid. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. A Second Pathway to Degrade Pyrimidine Nucleic Acid Precursors in Eukaryotes. The C-4 resonance of uracil shows a large coupling of 65 Hz to C-5 and a small coupling of 11 Hz to N-3. Identifications of mutations in strains NCM4139, NCM4299, NCM4300, and NCM4384.An 11-kbp deletion beginning in the mioC gene was first found in strain NCM4384 based on tiling microarray data from Roche Nimblegen (23). RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. 1 A upper pathway). During bubbling with O2, His6-RutA and Fre were added and bubbling was continued for an additional 5 min. The rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. 6). In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine‐5'‐monophosphate are directly catabolized by a reductive degradation pathway. 2B). The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The third product from uracil is β-alanine, and that from thymine is β-aminoisobutyric acid. This insertion occurred in a hot spot and is known to eliminate Lon protease activity (42). 4A). Apparently, they generate less than the usual amount of toxic malonic semialdehyde (see Results). We propose that it be called pyrimidine oxygenase. As was true of strains carrying rutA or rutF lesions, strains carrying a lesion in rutB in any of the three backgrounds we tested failed to grow on uridine as the sole nitrogen source (Table 5). Z-3-Ureido-2-propenoic acid (ureidoacrylate) was synthesized by adding 3 ml of 4 M ammonium hydroxide to 100 mg of 2,3-dihydro-1,3-6H-oxazine-2,6-dione (3-oxauracil) (Research Organics, Inc., Cleveland, OH) on ice as described previously (16). 2 However, some organisms, such as baker's yeast, Saccharomyces cerevisiae, cannot degrade pyrimidines at all. The NemA gene codes for the flavoprotein N-ethylmaleimide reductase, also referred to as the “old yellow enzyme” of E. coli (55). The pathway proceeds in three sequential enzymatic steps. The UpBCon1 strain had also acquired the deletion around mioC discussed above. The reductive pathway for pyrimidine degradation yields NH 3 and CO 2 from both uracil and thymine (Fig. We thank the National BioResource Project of the National Institute of Genetics, Japan, for E. coli Keio strains and ASKA strains. To confirm the identity of the RutA/F product, we chemically synthesized ureidoacrylate (Z-3-ureido-2-propenoic acid) from 3-oxauracil as described in Materials and Methods. The RutF protein was not well overexpressed and was assayed only in extracts. Genetic evidence indicates that RutE has the same function as YdfG: i.e., both reduce malonic semialdehyde to 3-hydroxypropionic acid, although presumably by different mechanisms. Comparison of Rut pathway products (E. coli K-12) to those of other pyrimidine catabolic pathways. (C) YdfG reaction. [14C-2]- and [14C-6]uracil were purchased from MP Biomedicals (Solon, OH). In vivo it yields 2 mol of utilizable nitrogen per mol of uracil or thymine and 1 mol of 3-hydroxypropionic acid or 2-methyl 3-hydroxypropionic acid, respectively, as a waste product (Fig. NMR chemical shifts of the RutA product are identical to those of synthetic ureidoacrylate, NMR chemical shifts and coupling constantsa of ureidoacrylate are identical to the published values. Evidence that RutE and YdfG have the same function. Moreover, a carbon-nitrogen HSQC spectrum on product labeled with 13C and 15N but not 18O indicated that N-3 had been converted to NH2 (see Fig. Copyright © 2020 Elsevier B.V. or its licensors or contributors. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. Hydrolytically to release 2 mol of ammonium would also inhibit spontaneous hydrolysis of the short-chain dehydrogenase (. Hydrolyze ureidoacrylate ( Fig were determined using the Micro bicinchoninic acid ( NH4+ salt 138. 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