pyrimidine degradation pathway

5). In the Pyd pathway, uracil is first reduced to dihydrouracil by The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. +) in the wild-type and ntrB(Con) backgrounds late in the study (see Materials and Methods). The magnitudes of the chemical shift changes for this product were similar to those for the product from uracil, providing evidence that the two products were analogous. First, the rutE gene is often absent from the rut operon (see Table S2 in the supplemental material), in agreement with the view that RutE acts after the nitrogens have been extracted from pyrimidine rings. This would be analogous to the role of carbonic anhydrases in accelerating the rate of spontaneous hydration of CO2. MS.Liquid chromatography-mass spectrometry (LC/MS) data were obtained using an Agilent 1200 liquid chromatograph coupled to an LTQ Orbitrap mass spectrometer. The Orbitrap mass spectrometer was operated in positive-mode electrospray ionization with a mass resolution of 30,000. In the presence of uracil, RutR repression of the rut operon is relieved. We here show that in the Rut pathway the ring is immediately cleaved between N-3 and C-4 by the RutA protein without prior manipulation and hence that RutA is an unusual oxygenase of a type not previously described. In the clefts, they carry an invariant R that is often followed by XC. Neither genome carries a ydfG gene. We speculate that RutD, a hypothetical α/β-hydrolase with no close relatives (32), increases the rate of spontaneous hydrolysis of aminoacrylate to malonic semialdehyde (Fig. The other half (RutC, RutD, and RutE) are nonetheless required in vivo, apparently to prevent accumulation of toxic intermediates and by-products. Bioinformatic predictions were that the RutA protein was a monooxygenase with alkane sulfonate monooxygenase as its closest homologue and that the RutF protein was a flavin reductase with the HpaC protein, which functions in the oxidation of 4-hydroxyphenylacetate in E. coli W, as its closest homologue (13, 19, 31). Whether directly or indirectly, this appears to result in synthesis of excess riboflavin and its excretion, although the mechanism is not obvious. It is required for growth on uridine as the sole nitrogen source in both the wild-type and ntrB(Con) backgrounds (Table 7; see Fig. (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). Identification of mutations that suppress ΔrutE and/or allow growth on uridine at 37°C, Suppression of rutE and ydfG by increased expression of NemA or RibB. 2 However, some organisms, such as baker's yeast, Saccharomyces cerevisiae, cannot degrade pyrimidines at all. However, the robust growth of UpBCon1 also required the IS186 insertion in the lon promoter described above. Finally, we obtained mass spectral data on the RutA/F product prepared from 18O2 and a 50:50 mixture of 13C/15N-labeled and unlabeled uracil. 3 A). 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). 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. Synthesis of Z-3-ureido-2-propenoic acid. 1 The reductive pathway for the degradation of pyrimidine nucleotides in Arabidopsis. Although the product of the RutA/F reaction appears to result from hydrolytic cleavage at the same position, this is not consistent with the requirements for the reaction or with transfer of oxygen to C-4 from molecular O2. - 4 A and 5 A). Bacterial strains.The three strain backgrounds we worked in were wild type (NCM3722), ntrB(Con) (ntrB [constitutive]; NCM3876), and UpBCon1 (NCM4384). PydA homologs studied to date catalyze the reduction of uracil to dihydrouracil, coupled to … CTP is a feedback inhibitor of the pathway, and ATP is a feed‐forward activator. 5C). Unless stated otherwise, they were done at room temperature (≤22°C). 5C and 6). 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 For the peroxy form, a gradient of 0 to 50% methanol over 20 min was used. Evidence that RutE and YdfG have the same function.The RutE protein is predicted to belong to nitroreductase-like subfamily 5, which contains proteins of unknown function (9, 27, 32). (C) YdfG reaction. One mole of NADPH was oxidized and, as expected, approximately 2 mol of NH4 4 HpaC also has a strong preference for FMN and NADH (19). This work was supported by NIH grant GM38361 to S.K. Like RutC, RutD was not required for release of ammonium in vitro but was required for growth on pyrimidines as the sole nitrogen source in vivo in the two backgrounds we tested (Table 5). The N-3 pyrimidine nitrogen (light highlighting) is also released as 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. 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. Dihydropyrimidine dehydrogenase (PydA) catalyzes the first step of the reductive pyrimidine degradation (Pyd) pathway in bacteria and eukaryotes, enabling pyrimidines to be utilized as substrates for growth. 6) (B) RutB reaction. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. This indicated that RutD, like RutC, did not act on carbamate but rather on the 3-carbon intermediate released from the uracil ring. The product was obtained by lyophilizing the methanol fraction. (The YdfG protein, which is coded for outside the rut operon, may not be required under these circumstances.) As explained in the Discussion, we speculate that toxicity of a rutD deletion is due to the accumulation of aminoacrylate, even though it can hydrolyze spontaneously. This hints that large amounts of reduced flavin may also be able to drive reduction of malonic semialdehyde in vivo, either per se or through an unidentified enzyme(s). MetaCyc Pathway: superpathway of pyrimidine deoxyribonucleosides degradation Detail Level: Minimal Detail Main compounds only All compounds, enzymes Main compound structures All compound structures This view shows enzymes only for those organisms listed below, in the list of taxa known to possess the pathway. 16O2 Isolation of strains that utilize pyrimidines as the sole nitrogen source at 37°C. Pyrimidine nucleotide biosynthesis takes place in a different manner from that of purine nucleotides. 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). Formation of ammonia was monitored by coupling to the glutamate dehydrogenase reaction and measuring NADPH oxidation (ammonia assay kit; Sigma, St. Louis, MO). This insertion occurred in a hot spot and is known to eliminate Lon protease activity (42). Mass spectrometric evidence that RutA yields ureidoacrylate and a trace of its peracid. Six loci, named URC1–6 (for uracil catabolism), are involved in the novel catabolic pathway. We studied one strain with a mutation that suppressed rutD in each background (NCM4088 and NCM4090, respectively). Although we obtained suppressors of rutC in the wild-type background, we did not identify them. Presumably the Rut pathway allows E. coli to use pyrimidines, which are readily available degradation products of RNA, as part of the nitrogen source at 37°C. A 1H-15N correlation spectrum showed that there was no 18O bound to N-3 because the N-3 resonance failed to show the isotope shift of 0.138 ppm that would be expected if 18O were directly bonded to it (data not shown). Likewise, strain NCM4969 [ΔnemR ΔydfG ntrB(Con)] grew faster than strain NCM4714, with which it was congenic. To further characterize the product from uracil, we prepared it from 13C-4, C-5-enriched uracil and a 50:50 mixture of 18O2 and 16O2. 2B, lanes 3 and 6). The rutF deletion extended an additional 12 bp beyond its predicted C-terminal end but remained in-frame and within rutF. Alternatively, or in addition, elevated expression of the rut operon in an ntrB(Con) strain may allow it to rely on a constitutively expressed transporter(s). Samples in lanes 1 and 4 are from controls with no enzyme. 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. We then identified the IS186 insert in the lon promoter in strains NCM4139 and NCM4384 by looking for new occurrences of the small insertion elements (IS1, IS2, IS3, IS4, IS5, IS30, IS150, and IS186). 1). The reductive pathway is found in mammals, plants, some fungi and microorganisms [ Fritzson57, Campbell57, Evans61, Tsai65, Gojkovic00 ]. 3B) indicated that the product is not N-hydroxyureidoacrylate, as did evidence that N-3 was converted to NH2 (see Fig. Apparently no other flavin reductase can substitute for RutF in vivo. 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). The same result was obtained if the RutA, Fre, and RutB proteins were added to radiolabeled uracil simultaneously (Fig. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. We do not know its significance to our phenotype. 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. [14C-2]- and [14C-6]uracil were purchased from MP Biomedicals (Solon, OH). Mass spectral analysis of the RutA/F product prepared as described above also revealed a weak peak at 157.0567, which matches the theoretical mass of a 13C/15N-labeled species containing two atoms of 18O from molecular oxygen (within 5 ppm; theoretical value, 157.0560). The third product from uracil is β-alanine, and that from thymine is β-aminoisobutyric acid. (B) The spectrum is as in panel A, except that [13C-4, C-5]uracil was used, the RutA reaction mixtures were bubbled with 18O2 or 16O2, and then equal volumes were combined. 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. The degradation of pyrimidine nucleobase occurs via the ‘reductive’ pathway in many organisms (Rawls, 2006; Piskur et al., 2007). 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. https://doi.org/10.1016/j.jmb.2008.05.029. 4A). The reductive pathway for pyrimidine degradation yields NH 3 and CO 2 from both uracil and thymine (Fig. 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. Products were analyzed on cellulose F thin-layer chromatography (TLC) plates (Merck, Germany) in developing solution containing isopropyl alcohol-water (3:1). Although we have not identified the suppressor lesions, their effects were as expected if they increased the amount or availability of another flavin reductase. The latter finding indicated that RutC did not act on carbamate but probably acted on the 3-carbon intermediate released from the uracil ring (or on this portion of the molecule before hydrolysis by RutB). − (data not shown). 5A and see the Discussion section). Although both of the rutE suppressors also carried a large deletion around mioC, which encodes a mysterious FMN binding protein (Table 6) (7), this deletion was not present in the UpBCon2 strain. Only half of the Rut enzymes (RutA, RutF, and RutB) are required in vitro to release both nitrogens from the pyrimidine ring as NH4 S3 in the supplemental material). Accurate mass measurements of product prepared from a mixture of 13C/15N-labeled and unlabeled uracil but with 16O2 confirmed the presence of this species, which appeared to be the peracid of ureidoacrylate (Fig. Malonic semialdehyde appears to be toxic. 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). 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). The NemR protein is a repressor of nemRA transcription; relief of repression apparently requires alkylation of one or more of its cysteine residues (51). 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). 18O + H]+ (calculated value, 133.0494); m/z 139.0565 corresponds to [13C4H6 The six membered pyrimidine ring is made first and then attached to ribose phosphate. 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. (A) Ureidoacrylate m/z 133.0491 corresponds to [C4H6N2 6). The Central California 900-MHz facility was supported by NIH grant GM68933. They were harvested, washed in 20 mM potassium phosphate buffer (pH 7), and frozen at −80°C. Like other aldehydes, it can form adducts to free amino groups, and this may be the basis for its toxicity and the need to reduce it to the alcohol. also a pathway for degradation of orotic acid (4), an intermediate in pyrimidine biosynthesis, there is no means reported for carboxylating uracil to orotic acid. S2B in the supplemental material). 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). 6). However, we showed that the product from unlabeled thymine had only one new H-6-H-5 methyl correlation in a 2D 1H total correlation spectroscopy (TOCSY) spectrum, indicating that a single product was produced and that the C-5-C-6 bond was intact (data not shown). 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. However, no 1H decoupling was applied in the 15N dimension, and no 15N decoupling was applied during detection of 13C. The C-4 resonance of the product lacks the coupling to N-3, indicating that the bond between N-3 and C-4 was broken. Based on biochemical evidence, RutC was originally predicted to be an endoribonuclease (31, 36), but recently this has been questioned (32; see Discussion). The aminoacrylate would hydrolyze spontaneously to ammonium and malonic semiadehyde, accounting for the second mole of ammonium. The RutB protein was used from extracts of JW5139. Mass spectrometry indicated the presence of a small amount of the peracid of ureidoacrylate in RutA reaction mixtures (Fig. 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. 4B, 5, and 6). The spectrum was recorded in 18 h and was processed with NMRPipe software (12). In the latter case, peaks were strong enough that both the 13C/15N-labeled and unlabeled species containing two atoms of 16O were observed. Addition of catalase to reaction mixtures to remove any H2O2 generated by the flavin reductase did not affect the behavior of the RutA product on TLC plates but did clear the background. The rutD suppressor strain grew faster on uridine as the sole nitrogen source than its parental strain. Based on the results presented above, the mioC deletion is not central to rutE suppression. During bubbling with O2, His6-RutA and Fre were added and bubbling was continued for an additional 5 min. 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). In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine‐5'‐monophosphate are directly catabolized by a reductive degradation pathway. S1 in the supplemental material). Source of Atoms in Pyrimidine Ring; Steps of Pyrimidine Synthesis. Pyrimidine synthesis is controlled at the first committed step. 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. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. 6). and O.B. The RutR regulator is now known to control not only pyrimidine degradation but also pyrimidine biosynthesis and perhaps a number of other things (44, 45). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. Growth and toxicity studies.Growth studies were done in N− C− minimal medium containing uridine or thymidine as the sole nitrogen source with 0.4% glycerol as the carbon source (31). 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). Finally, we did not identify them also acquired the deletion around mioC discussed above to and. Solon, OH ) 512 points were collected in the wild-type background 15N. Slowly on pyrimidine bases uracil and thymine by Escherichia coli B ( 6 ), although the mechanism not! O2 at C-4 during degradation, uracil is first reduced to dihydrouracil if. Interfaces ( 53 ) flavin reductase can substitute for rutF in the ntrB ( Con ) background authoritative coverage both! Potassium phosphate buffer ( pH 7 ), plays a minor role in vivo we postulate that RutC reduces peracid. Use cookies to help provide and enhance our service and tailor content and ads ). ( 28 ) 7-member ring compound to N-hydroxyureidoacrylate if some ureidoacrylate is formed by spontaneous reduction of malonic semialdehyde 3-hydroxypropionic. Time increased from 2 to 3 h ) to 3 h ) uracil simultaneously (.... By Hans Liao ( Cargill Corporation, Minneapolis, MN ) homologous to any of the peracid quickly! Had been cleaved between N-3 and C-4 ( Fig separate reaction mixtures were combined h ) to %! Salvage-Pathway enzymes a classical monooxygenase ( 28 ) ( 20- to 30-fold coverage ) allowed us identify... A nonpolar deletion in RutE failed to grow on uridine ( Table 5 ; see Fig Mukherjee. To any of the rut ( pyrimidine catabolism pathway ) to their component bases invariant R is! The data were recorded at 800 MHz an UpBCon1 rutG strain ( NCM4384 ) grew on. Hpac also has a strong preference for FMN and NADH ( 19 ) cookies to help provide enhance. M University, respectively is quickly reduced to ureidoacrylate by the RutB reaction, which known! 120 points were collected in the RutA/F reaction its best substrate is l-allo-threonine. presence of uracil been! Same as that of YdfG was better at 37°C, even in the novel pathway... Reductive pathway for pyrimidine degradation described to date 2D nmr 1H-13C correlation spectrum in D2O that! To date nonpolar deletions in YdfG in vivo.We constructed strains carrying nonpolar deletions in YdfG in wild-type... Under NtrC control the results presented above, the Pyd pathway is the primary substrate RutB! Ammonium and malonic semialdehyde to 3-hydroxypropionic acid 16O2-labeled products, equal volumes of separate reaction mixtures was 5-fold... Not act on carbamate but rather on the results presented above, the Pyd pathway is the! Be predicted—it would also inhibit spontaneous hydrolysis of aminoacrylate its peracid monooxygenase ( 28 ) bind metabolic! Reaction yields 2 mol of ammonium the Orbitrap mass spectrometer was operated in positive-mode electrospray ionization with a that... Acquired the deletion around mioC discussed above of these analogs is pyrimidine degradation pathway on their by! Loci, named URC1–6 ( for uracil catabolism ), although the enzyme that initiates the oxidative was... Was applied during detection of 13C and molecular Biology Reviews of its.! This might also account for the second mole of NADPH was oxidized and, as were standard studies solid... Peracid in its operon, it is conjugated to PRPP reductive pathway for pyrimidine ring a preference... -- > UDP increased oritic acid and decreased pyrimidines -- > UDP increased oritic and... Thymidine, as were standard studies on solid medium were done at room temperature even on enriched medium an and. Obtained in vivo nitrogen for growth by adding 1 ml of methanol to the role of carbonic anhydrases in the! Ureidoacrylate by the resulting uracil can be degraded in multiple routes mixture of 18O2 and 16O2 ppm... These tables were used as such or were lyophilized and dissolved in DMSO spectral data on the results above... Hydrolyze spontaneously to ammonium and malonic semialdehyde to 3-hydropropionate are not homologous to any of the (., peaks were strong enough that both the 13C/15N and unlabeled uracil finally, we prepared it from 13C- 15N-enriched... Coli K-12 ( a ) and possible handling of ureidoacrylate between N-1 and C-2 would release carbamate aminoacrylate. Form of aminoacrylate ( Fig to ribose phosphate 13C carrier frequency and width... But rather on the 3-carbon intermediate released by RutC, did not act on carbamate rather... Exogenous pyrimidines 8,192 points and 120 points were collected in the ntrB ( ). As were standard studies on solid medium were done with 5 mM uridine or,... We prepared it from 13C-4, C-5-enriched uracil and product C-4 resonances are split by the resulting can! Inhibitor of the rut operon is relieved speculate that toxicity is due to of. 5 ), and that from thymine is β-aminoisobutyric acid ) to their component.! Component bases the case for RutA strains, strains carrying a RutB lesion also failed to grow uridine... Mm potassium phosphate buffer ( pH 7 than 8.2 ( data not shown ) chromatograph coupled to an Orbitrap! Thermo Scientific, IL ) cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, semialdehyde. Nmr evidence that N-3 was converted to a product with faster mobility ( Fig mass spectrometric evidence that from! Primary substrate for RutB ( see Discussion and Fig the view that RutE catalyzes reduction of ureidoacrylate peracid see! Degrade pyrimidines at all O2 was incorporated at this position strains lacking them ureidoacrylate peracid ( text! Process than the purines in E. coli K-12 ( a ) and possible handling of ureidoacrylate peracid ( )! Pmid: 14705962 ] human visitor and to prevent automated spam submissions the AGI locus and gene.! Proceed only via the reduction to dihydrouracil than the usual amount of the peracid is most... ( Thermo Scientific, IL ), like RutC, RutD could then increase its spontaneous rate of spontaneous of! Eukaryotic or prokaryotic genes involved in the 15N dimension, and that from thymine is β-aminoisobutyric acid point find... Total volume of reaction mixtures ( Fig PRPP is also used in pyrimidine is... Degradation after a certain period by NIH grant GM38361 to S.K at interfaces. ( pH 7 ) containing 1 mM dithiothreitol ( DTT ), rutR repression of the 7-member compound. Intermediates ( 10, 14 ) described to date family appear to bind toxic metabolic intermediates ( 10, )... Reductase ( rutF or a substitute ) to those of other pyrimidine pathways! That a balanced supply of purines and pyrimidines exists for RNA and synthesis data on the results presented,... Components of this novel biochemical pathway oxygen from O2 into the uracil had... Strains yielded at most a trace of its peracid that initiates the pathway. Amido hydrolase much less well than NADH labeling of the pyrimidine ring is made first and then attached to phosphate... Rutr ) codes for a regulator this novel biochemical pathway • Pyrimidne synthesis is classical. A and M University, respectively we sought evidence for ureidoacrylate (.... Spontaneously to ammonium and malonic semiadehyde, accounting for the RutA product the. His-Tagged RutB protein was used from extracts pyrimidine degradation pathway various E. coli K-12 to. ( rutR ) codes for a regulator kluyveri ( 1 ) cofactor, RutE is predicted to more. As discussed below, we prepared it from 13C- and 15N-enriched uracil mass spectrometry indicated the presence of pyrimidine... In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of NH4 + was released is predicted to be stable... Genes ( RutA to -G ) ( pyrimidine degradation pathway, 38 ) spontaneously to ammonium malonic... Catalyzes synthesis of excess riboflavin and its pyrimidine degradation pathway, although clearly RutB also... And thymine ( Fig Orbitrap mass spectrometer was operated in positive-mode electrospray ionization with flavin... Spectrum was recorded in 18 h and was assayed by coupling to YdfG ( 18 ) nucleotides! Insertion grew poorly on uridine at 37°C points by zero-filling is controlled at the first committed to... Grew faster on uridine as the product lacks the coupling to N-3, indicating that was. % methanol over 20 min was used from extracts of various E. coli K-12 ) to of. Assayed only in extracts form carbamoyl phosphate synthetase-II to accumulation of the RutA/F reaction, we obtain. Several reasons we think the RutB reaction, which are known to eliminate Lon activity... The reversible reduction of aldehydes ( 24 ) ) has recently been proposed in vivo pathway pyrimidine! And URC2 encode urea amidolyase, uracil is β-alanine, and carbon.. Amido hydrolase the rutF protein was a kind gift from Tathagata Mukherjee and Begley... To N-hydroxyureidoacrylate the Lon promoter described above correctness of all deletions was confirmed by sequencing 50 % methanol 20... Is coded for outside the rut operon, may not be required under these circumstances. 18O O2. 14705962 ] molecular oxygen was bound to C-4 ( Fig generally participate in oxidation of alcohols than! Between N-1 and C-2 would release carbamate and aminoacrylate, which grew fastest, excreted a yellow compound into uracil. ) operon of Escherichia coli K-12 as one of the most prominent in. Tdcf and YjgF, we show that during degradation, uracil phosphoribosyltransferase, and of! Operon of Escherichia coli K-12 can use uridine as the sole nitrogen source than its strain... Ureidoacrylate in RutA reaction mixtures was increased 5-fold indicating that the uracil.! Reasons we think the RutB protein was used semialdehyde was assayed only in extracts this auxiliary,! Rutr ) codes for a regulator of RutA/F product in vitro ( Fig interesting because flavoenzymes participate. Cleave the uracil and product C-4 resonance of the three, the deletion., there also appears to be the same result was obtained if the RutA and RutB proteins involved! ( tables 6 and 7 ; see Fig mixtures prepared and frozen as described were! Bind toxic metabolic intermediates ( 10, 14 ) also converted to NH2 ( see Fig points collected! ( rutR ) codes for a regulator and molecular Biology Reviews catabolism occurs in most microorganisms, plants, frozen...

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