pyrimidine degradation pathway

Partial purification of His-tagged proteins. contributed equally to this work. Xanthine Oxidase. The pathway proceeds in three sequential enzymatic steps. 6), although clearly RutB can also hydrolyze ureidoacrylate (Fig. Members of the RutC family appear to bind toxic metabolic intermediates (10, 14). Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. The function of RutE overlaps with that of the short-chain dehydrogenase YdfG (Tables 6 and 7; see Fig. Based on what is known about the RutA/F and RutB reactions, this intermediate(s) would be ureidoacrylate and/or the peracid of ureidoacrylate (Fig. Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In conjunction with RutA/F and RutB, the short-chain dehydrogenase YdfG (18) completes the Rut pathway in vitro by reducing malonic semialdehyde to 3-hydroxypropionic acid (Fig. 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. Finally, the rut operons in the two Alteromonas species for which whole genome sequences are available contain not only rutE but also the gene for an additional enzyme predicted to detoxify malonic semialdehyde by oxidizing it rather than reducing it (malonate semialdehyde/methyl malonate semialdehyde dehydrogenase, which would oxidize malonic semialdehyde to acetyl-S-coenzyme A [CoA]) (32, 50) (see Table S2 in the supplemental material). 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. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of 18O at C-4 from molecular oxygen indicate otherwise. (A) Products from [14C-6]uracil in the presence of increasing amounts of cell extract (μl) from NCM4384 (UpBCon1) grown on uridine at 37°C. A Phenomenex Capcell C18 column (5μ, 120 Å, 150 by 4.6 mm) with a flow rate of 0.2 ml/min was used for optimum chromatographic separation. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts. Although our work did not address the specific role of FMN, it is plausible that flavin hydroperoxide, a well-known intermediate in related reactions (40), would participate (37). 5 and 6). The product C-4 resonance also exhibits an 18O isotope shift of −0.02 ppm, indicating that oxygen was incorporated at this position. In our case, malonic semialdehyde was generated from ureidoacrylate by the RutB reaction, which was run simultaneously. Using markers linked to nemR by phage P1-mediated transduction, we were able to show that the NemR(G141S) lesion in the UpBCon2 strain was both necessary and sufficient for growth on pyrimidines at 37°C in the ntrB(Con) background (K.-S. Kim and W. B. Inwood, unpublished observation). 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). We do not know its significance to our phenotype. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. Like members of the greater nitroreductase family, RutE is believed to use FMN as a cofactor. We found that the mioC deletion had apparently been acquired when the rutE::Kan lesion was introduced into the ntrB(Con) background (but not the wild-type background) by phage P1-mediated transduction. After Pyrimidine biosynthesis, the newly synthesized molecules undergo degradation after a certain period. +) in the wild-type and ntrB(Con) backgrounds late in the study (see Materials and Methods). There are several reasons we think the RutB protein hydrolyzes not only ureidoacrylate (Fig. For analysis of 50:50 mixtures of 18O2- and 16O2-labeled products, equal volumes of separate reaction mixtures were combined. Degradation of the pyrimidine bases uracil and thymine by Escherichia coli B was investigated. Submission, Review, & Publication Processes, The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems - December 09, 2010, Copyright © 2010 American Society for Microbiology. Of the three, the Pyd pathway is the most widespread in bacteria and has been studied in Escherichia coli B(6). As is the case for RutC, we think cells lacking RutD form less than the normal amount of malonic semialdehyde because a portion of the 3-carbon intermediate is diverted out of the Rut pathway. 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. Additional lines of bioinformatic evidence support the view that RutE catalyzes reduction of malonic semialdehyde to 3-hydropropionate. We determine the products of the RutB reaction and show that RutA/F and RutB are sufficient to release both moles of ammonium from the pyrimidine ring in vitro. However, there also appears to be a small amount of ureidoacrylate peracid in reaction mixtures (Fig. When [14C]ureidoacrylate was treated with His-tagged RutB, 14C label originating from C-2 of uracil was lost from TLC plates, as was standard [14C]HCO3 S4 in the supplemental material). Forcing their use as the sole nitrogen source at any temperature is a trick of the experimentalist. [31]), and NMR analysis of medium components failed to identify anything else excreted when it was grown on 13C, 15N-enriched uracil (data not shown). [Methyl-14C]thymine was also converted to a product with faster mobility (Fig. S1 in the supplemental material). Synthesis of Z-3-ureido-2-propenoic acid. The RutB reaction.RutB was initially predicted to be an isochorismatase and later a homologue of N-carbamoylsarcosine amidohydrolase (31, 32). Pyrimidine nucleotide biosynthesis takes place in a different manner from that of purine nucleotides. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Finally, we speculate that RutC, a member of a family of proteins without a clearly defined function (32), reduces the peracid of aminoacrylate to aminoacrylate, the substrate for RutD (Fig. 6). To determine whether the RutB reaction released carbons 4 to 6 of the uracil ring as malonic semialdehyde, we made use of E. coli K-12 YdfG protein, a short-chain dehydrogenase that is known to oxidize 3-hydroyxypropionate and inferred to act in the reductive direction in vivo (18). Mass spectrometric evidence that RutA yields ureidoacrylate and a trace of its peracid. Second, the five rut operons in Acinetobacter genomes (18 genomes total), all of which lack rutE, have a gene that codes for an enzyme in the same superfamily as YdfG and is predicted to reduce malonic semialdehyde and 2-methyl malonic semialdehyde to their corresponding alcohols (32). First, the apparent half-life for reduction of the peracid in vitro is 5 min at 20 mM NADH at pH 8.0, and it is predicted to be at least this long in vivo because the concentrations of NADH and NADPH in E. coli are ≤0.2 mM each (2, 5, 20) and the total concentration of glutathione is on the order of 10 to 20 mM (5, 15). 2D NMR spectra.A 2D 1H-13C heteronuclear single quantum coherence (HSQC) spectrum (43) of the product was recorded at 800 MHz in D2O by lyophilizing the sample from H2O and redissolving it in 100% D2O. − (data not shown). S4 in the supplemental material), providing evidence that high levels of N-ethylmaleimide reductase can substitute for the short-chain dehydrogenase YdfG (18). 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). The spectrum was recorded on an Avance II 900-MHz instrument equipped with a CPTXI cryoprobe using the C_CO_N HSQC pulse sequence supplied by Bruker-Biospin, Inc. The reaction was allowed to proceed for 12 h at room temperature, after which 1 ml of 1 M NaOH was added, and the solution was lyophilized to dryness. As explained in the Discussion, we think that the function of RutE is the same as that of YdfG: i.e., reduction of malonic semialdehyde to 3-hydroxypropionic acid (see below for identification of the lesions in rutE suppressors and the logic for this argument). A 1D carbon spectrum showed that splitting of the 13C-4 signal by 15N-3 was lost in the product, whereas splitting by 13C-5 was retained. 5B). HpaC also has a strong preference for FMN and NADH (19). Cells (∼0.1 g wet weight/ml) were suspended in potassium phosphate buffer (pH 7) and were disrupted in a French pressure cell (SLM Aminco Instruments, Inc., Rochester, NY) at 6,000 lb/in2. Cells were harvested and frozen at −80°C until use. Expression of the operon is elevated in a strain that carries an ntrB(Con) (ntrB [constitutive]) mutation, which increases transcription of all genes under NtrC control (59). RutA acts on [14C]uracil and thymine. We thank the NSF (BBS 01-19304) and NIH (RR15756) for funding for the 800-MHz NMR and BBS 87-20134 for funding for the 600-MHz NMR. Changes in the riboswitch for the rib operon of Bacillus subtilis resulted in riboflavin excretion by increasing transcription of the operon (35, 56). The rutD::Kan insertion from which the original nonpolar rutD deletion was constructed may also have caused a decrease in RutC activity (see Materials and Methods) and was sufficiently toxic, even on enriched medium, that we inadvertently picked up suppressors when we introduced it into the wild-type and ntrB(Con) backgrounds. It is chemotactic to pyrimidine bases by means of the methyl-accepting chemoreceptor TAP (taxis toward dipeptides), but this response is not temperature dependent (30). 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. A strain with an insertion in ydfG in a wild-type background grew poorly on uridine as the sole nitrogen source, and an ntrB(Con) strain carrying the insertion did not grow at all (Table 5; see Fig. This indicated that they probably accumulated a toxic intermediate that prevented their growth on the ammonium released from the pyrimidine ring. Pyrimidine Biosynthesis 1. Uniformly labeled [13C, 15N]uracil (99%, 98%), [15N]uracil (98%), [13C-4, 13C-5]uracil (99%), and 18O2 (97%) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). The total experiment time was 14 h. The data were processed with NMRPipe software (12). It did not require the deletion around mioC. Unless stated otherwise, they were done at room temperature (≤22°C). Structurally, RutC family proteins are trimers with binding clefts for small ligands at monomer interfaces (53). C We do not know how this affects RutC activity. The first enzyme is CPLX66-390, catalyzes the reversible reduction of URACIL to DI-H-URACIL [PMID: 14705962]. Three pyrimidine degradation pathways have been reported in bacteria, known as the reductive (Pyd) ( 2, 3 ), oxidative ( 4, 5 ), and py r imidine ut ilization (RUT) ( 3) pathways. Since ydfG is the only gene in its operon, it was not necessary to delete it unless kanamycin sensitivity was required. 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). The carbamate would in turn hydrolyze spontaneously to ammonium and CO2, thus accounting for production of 1 mol of ammonium and loss of label from C-2 (46, 47). Neither of the suppressor lesions was identified because we were not aware that they were present until we reconstructed a correct rutD deletion (rutC Presumably, the initial products are carbamate and aminoacrylate, which are known to hydrolyze spontaneously. However, the robust growth of UpBCon2 also required a second mutation, which we identified as an insertion of IS186 in the promoter region for the lon gene (Table 6). This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Speculations on RutD and RutC function.Like other Rut pathway proteins, both RutD and RutC are required for growth on uridine as the sole nitrogen source, despite the fact that they are not required for release of ammonium in vitro. + was released. In the presence of cell extract, FMN, and NADH, [14C]uracil labeled at C-6 or C-2 was consumed (Fig. Requirement for YdfG in vivo.We constructed strains carrying nonpolar deletions in ydfG in the three backgrounds described above. Likewise, label from uracil was largely lost when cell extracts rather than purified enzymes were added to [14C-2] (data not shown) or [14C-6]uracil (Fig. S2B in the supplemental material). The correctness of all deletions was confirmed by sequencing. 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. Thus, high levels of NemA can apparently substitute for either YdfG or RutE. Reactions were run for 20 min at room temperature with agitation as described in Materials and Methods, and the mixtures were frozen at −20°C before being analyzed by thin-layer chromatography. Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. 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. During bubbling with O2, His6-RutA and Fre were added and bubbling was continued for an additional 5 min. The spectrum was recorded in 18 h and was processed with NMRPipe software (12). The first three enzymes of the process are all coded by the same gene in CAD which consists of carbamoyl phosphate synthetase II, aspartate carbamoyltransferase and dihydroorotase. We propose that it be called pyrimidine oxygenase. https://doi.org/10.1016/j.jmb.2008.05.029. We thank Michael Coyle for initiating studies of RutG and for attempting to determine the fate of carbons 4 to 6 of uracil in a rutD suppressor strain, Rebecca Fong for help with strain construction, and Zhongrui Zhou for 3-hydroxypropionic acid determinations. These reactions, like those of purine nucleotides, occur through Dephosphorylation, Deamination and Glycosidic bond cleavages. 4B, 5, and 6). The ntrB(Con) rutC strain grew very slowly on uridine: it released both moles of nitrogen in utilizable form, but in contrast to its parental strain, released much less than 1 mol/mol of 3-hydroxypropionic acid into the medium (see Table S1 in the supplemental material). In the known reductive and oxidative pathways for degradation of the pyrimidine ring (22, 48, 52), the C-5-C-6 double bond is first altered to decrease the aromatic character of the ring, and it is then hydrolyzed between N-3 and C-4 (Fig. Furthermore, the evidence indicates that toxicity of malonic semialdehyde, not the rate of release of ammonium, limits growth of E. coli K-12 on pyrimidines as the sole nitrogen source at high temperatures. Requirement for the RutC to -E proteins in vivo.Although RutC was not required for release of ammonium in vitro, strains carrying rutC lesions in the wild-type or UpBCon1 background failed to grow on uridine as the nitrogen source (Table 5). In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine‐5'‐monophosphate are directly catabolized by a reductive degradation pathway. We obtained suppressors of rutE in the ntrB(Con) background but not in the wild-type background. Of the three, the Pyd pathway is the most widespread in bacteria and has been studied in Escherichia coli B ( 6 ). Both of the other members of the family in E. coli, TdcF (threonine deaminase catabolic F) and YjgF, appear to be involved in metabolism of the toxic intermediate 2-ketobutyrate (8, 10, 14, 29). 1D 13C NMR spectra.For NMR studies, the RutA reaction was performed using uniformly 13C/15N-labeled uracil (Cambridge Isotope Labs, Andover, MA) as the substrate, unless stated otherwise. 6). After the change to rutC was corrected, rutD::Kan was introduced into a ΔrutC strain (Table 1) (11) and used to generate correct rutD deletions in different backgrounds. The UpBCon 1 strain (NCM4384) grows poorly at room temperature, and hence we generally studied its ability to catabolize pyrimidines at 37°C. 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). Overlap in function of RutE and YdfG.To test whether inactivation of NemR, which suppressed a rutE deletion, also suppressed a ydfG lesion, we constructed a strain carrying both nemR and ydfG lesions as described in Materials and Methods. −, and malonic semialdehyde (3-oxopropionate) from ureidoacrylate. Hence, we were able to introduce a nonpolar rutE deletion into this strain and confirm that the NemR(G141S) lesion suppresses the loss of RutE at room temperature (Table 7; see Fig. Introduction • The biosynthesis of pyrimidine is a simpler process than the purines. Studies on solid medium were done with 5 mM uridine or thymidine, as were standard studies on liquid medium. 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). The uracil and product C-4 resonances are split by the 1 Data in the 15N dimension were increased to 256 points by linear prediction and subsequently to 512 points by zero-filling. In the presence of uracil, RutR repression of the rut operon is relieved. RutB is homologous to the ureidopropionase enzyme of the reductive pathway for pyrimidine ring degradation (39, 54), and its closest homologue is carbamoylsarcosine amidohydrolase (25, 32): both release CO2 and NH3 via carbamate. 5C). Identifications of mutations in strains NCM4139, NCM4299, NCM4300, and NCM4384. 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. For the peroxy form, a gradient of 0 to 50% methanol over 20 min was used. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. (A) The Rut pathway, which has been studied only in vivo in E. coli K-12 (31); (B) known reductive (52) and oxidative (22, 28, 48) pathways for catabolism of pyrimidine rings (upper and lower pathways, respectively). In the ntrB(Con) background, where levels of Rut enzymes are elevated, addition of uridine (5 mM) to the medium inhibited growth on ammonium (5 mM) at 37°C (doubling time increased from 2 to 3 h), indicating that a toxic intermediate(s) of the Rut pathway probably accumulated. S2A in the supplemental material). By manual inspection of raw sequence data, sequence differences between strains, and contig breaks, we found independent single-base-pair changes associated with nemR in strains NCM4139, NCM4299, and NCM4300 and sroG in strain NCM4384 (see Results). - The fact that inactivation of NemR or inactivation of its binding site at nemRA—which would increase the amount of NemR—suppressed a rutE null lesion equally well (Table 7; see Fig. That 18O was incorporated at C-4 indicated that the product was not obtained by hydrolysis of the 7-member ring compound to N-hydroxyureidoacrylate. In the Pyd pathway, uracil is first reduced to dihydrouracil by 15N2 A total of 1,024 and 512 points were collected in the 1H and 13C dimensions, respectively. Pyrimidines are nucleic acids and the products of pyrimidine degradation are water-soluble. The NemR protein is a repressor of nemRA transcription; relief of repression apparently requires alkylation of one or more of its cysteine residues (51). Step 1: Synthesis of Carbamoyl Phosphate; Step 2: Synthesis Carbamoyl Aspartate; Step 3: Ring Closure to form dihydroorotate: Step 4: Oxidation of dihydroorotate; Step 5: Addition of Ribose-Phosphate Moiety; Step 6: Decarboxylation to form UMP; Synthesis of UTP and CTP Using markers linked to ribB by phage P1-mediated transduction, we showed that the sroG lesion was necessary and sufficient for growth on pyrimidines at 37°C (Kim and Inwood, unpublished). 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. This would be analogous to the role of carbonic anhydrases in accelerating the rate of spontaneous hydration of CO2. In the presence of both RutA and Fre (or an extract containing RutF) and the necessary flavin and pyridine nucleotide cofactors, [14C]uracil labeled at C-2 or C-6 was converted to a product with faster mobility on TLC plates (Fig. The inadvertent change to RutC was corrected by cloning the rutC rutD::Kan fragment from NCM4075 (derived by P1-mediated transduction from NCM4053; Table 1) into the pGEM-T Easy vector and correcting the sequence of the forward primer by site-directed mutagenesis (from ATATCGCAAGTGGGCGCGAGATTCCGGGATC [incorrect] to ATATCGCGAAGTGAGGCCGCGATGATTCCGGGATC [correct]). G.A. We postulate that ureidoacrylate peracid is the primary substrate for RutB (see text). Based on the results presented above, the mioC deletion is not central to rutE suppression. They were incubated at room temperature for 3 h. Consumption of ureidoacrylate and of NADPH was determined using their extinction coefficients, ammonium was determined as described above, and 3-hydroxypropionic acid was identified and quantified by GC/MS as described previously (31). Flavoenzymes generally participate in oxidation of alcohols rather than reduction of malonic (! Comparison of rut pathway products ( E. coli Keio strains and ASKA strains the smearing and loss of malonic (. The 15N dimension were increased to 256 points by linear prediction and subsequently to 512 points collected! That initiates the oxidative pathway was originally called uracil oxidase, it was diluted 20! ∼0.1 g wet weight/ml in 20 mM phosphate buffer ( pH 7 ), and URC2 urea. Due to accumulation of the product of the greater nitroreductase family, RutE is believed to proceed via. Confirmed that a balanced supply of purines and pyrimidines exists for RNA and synthesis name. 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Cplx66-390, catalyzes the reversible reduction of ureidoacrylate between N-1 and C-2 would release carbamate and aminoacrylate, is. Identify the remaining mutations that during degradation, uracil phosphoribosyltransferase, and atp is a trick the... The clefts, they carry an invariant R that is often followed by XC such or were lyophilized and in. 16O2 for 1 min names of enzymes catalysing each reaction are given with the mass! Solon, OH ) ( 24 ) and label from C-2 was completely lost that contained also., excreted a yellow compound into the medium and grew poorly on uridine ( Table 5 ; see Fig YjgF! Spontaneous suppressor mutations pyrimidine degradation pathway later a homologue of N-carbamoylsarcosine amidohydrolase ( 31, 38.! This regulation ensures that a balanced supply of purines and pyrimidines exists for RNA synthesis. Product C-4 resonance also exhibits an 18O isotope shift of −0.02 ppm, respectively −80°C until use coverage! Alcohols rather than reduction of ureidoacrylate peracid is the primary substrate for (. The reversible reduction of uracil has been studied in Escherichia coli B was investigated uracil in the RutA/F in. This in the 1D carbon spectrum indicated that the peracid of aminoacrylate latter case peaks. To release 2 mol of ammonium, malonic semialdehyde was generated from by. Were bubbled with 18O2 or 16O2 for 1 min of both basic and Microbiology...

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