Of the three, the Pyd pathway is the most widespread in bacteria and has been studied in Escherichia coli B(6). The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. S2B in the supplemental material). 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. Samples in lanes 1 and 4 are from controls with no enzyme. If reduction of the peracid in vivo is slow, some spontaneous decomposition—to ureidoacrylate, uracil, and undefined by-products (37)—could occur if RutB did not hydrolyze the peracid rapidly. Evidence that RutE and YdfG have the same function. For analysis of 50:50 mixtures of 18O2- and 16O2-labeled products, equal volumes of separate reaction mixtures were combined. Growth inhibition persisted in an ntrB(Con) rutF strain (doubling time increased from 2 to 3 h). In conjunction with a flavin reductase, RutA uses molecular oxygen to cleave the uracil ring between N-3 and C-4 (Fig. [14C-methyl]thymine was purchased from Moravek Biochemicals and Radiochemicals (Brea, CA). 2B). Source of Atoms in Pyrimidine Ring; Steps of Pyrimidine Synthesis. (Lactic acid is 2-hydroxypropionic acid.) The UpBCon1 strain carrying a ydfG insertion grew poorly on uridine at 37°C. 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). 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. 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). 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. 6). 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. 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. Isolation of strains that utilize pyrimidines as the sole nitrogen source at 37°C. In the Pyd pathway, uracil is first reduced to dihydrouracil by For 18O2 labeling of the RutA product, the total volume of reaction mixtures was increased 5-fold. 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. The correctness of all deletions was confirmed by sequencing. [14C-2]- and [14C-6]uracil were purchased from MP Biomedicals (Solon, OH). The reductive pathway is found in mammals, plants, some fungi and microorganisms [ Fritzson57, Campbell57, Evans61, Tsai65, Gojkovic00 ]. 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). 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). 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. Here, we identify two Arabidopsis ( Arabidopsis thaliana ) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. The resulting uracil can be degraded in multiple routes. 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. 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). 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 reductive pathway for pyrimidine degradation yields NH 3 and CO 2 from both uracil and thymine (Fig. 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). 6), although clearly RutB can also hydrolyze ureidoacrylate (Fig. 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). Hydrolytic cleavage of ureidoacrylate between N-1 and C-2 would release carbamate and aminoacrylate (Fig. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. CTP is a feedback inhibitor of the pathway, and ATP is a feed‐forward activator. 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). Since ydfG is the only gene in its operon, it was not necessary to delete it unless kanamycin sensitivity was required. 15N2 After zero-filling twice in the 13C dimension, the digital resolution was 16 Hz/point. 3B) indicated that the product is not N-hydroxyureidoacrylate, as did evidence that N-3 was converted to NH2 (see Fig. At 37°C, it obtained both nitrogens from uridine in usable form and excreted 1 mol/mol of 3-hydroxypropionic acid into the medium (see Table S1 in the supplemental material) (31). Wild-type or ntrB(Con) strains with a nonpolar deletion in rutE failed to grow on uridine (Table 5; see Fig. C The rutF deletion extended an additional 12 bp beyond its predicted C-terminal end but remained in-frame and within rutF. 2B, lanes 3 and 6). The pathway proceeds in three sequential enzymatic steps. − (data not shown). 4A). 1 The reductive pathway for the degradation of pyrimidine nucleotides in Arabidopsis. 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). Based on the results presented above, the mioC deletion is not central to rutE suppression. 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). The structure of the E. coli TdcF protein has been determined with 2-ketobutyrate bound: oddly, it was bound as the rare enol tautomer, with its carboxylate group doubly hydrogen bonded to the guanidinium group of the invariant R and its enol OH group bonded to both the backbone amide of the conserved C and the carboxyl group of the conserved E120 in the adjacent subunit (8) (see Fig. We thank Hans Liao for the gift of 3-hydroxypropionic acid and for alerting us to the possible role of the YdfG protein in its formation, and we thank Chris Walsh for alerting us to a mechanism by which RutD could increase the rate of aminoacrylate hydrolysis. The reasoning for these conclusions is as follows. S4 in the supplemental material). Malonic semialdehyde appears to be toxic. 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. The salvage pathways of pyrimidine ribonucleotides consist of 1) importing exogenous bases into the cell, and 2) the interconversion of various bases (CITS:2189783)(CITS:12111094). Using E. coli K-12 strain MG1655 as a reference strain for the assembly of each of the four genomes, Roche provided tables of differences between each strain and MG1655. The first enzyme is CPLX66-390, catalyzes the reversible reduction of URACIL to DI-H-URACIL [PMID: 14705962]. As was the case for rutA strains, strains carrying a rutB lesion also failed to yield spontaneous suppressor mutations. S2B in the supplemental material). 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. A total of 1,024 and 512 points were collected in the 1H and 13C dimensions, respectively. All of the findings were in agreement with ureidoacrylate as the product. RutG appears to be a uracil transporter. Although we have not identified the suppressor lesions, their effects were as expected if they increased the amount or availability of another flavin reductase. Three pyrimidine degradation pathways have been reported in bacteria, known as the reductive (Pyd) (2, 3), oxidative (4, 5), and pyrimidine utilization (RUT) (3) pathways. The carrier frequencies were set to 170 ppm (13C) and 95 ppm (15N), and spectral widths were set to 80 ppm (13C) and 87.5 ppm (15N). Forcing their use as the sole nitrogen source at any temperature is a trick of the experimentalist. 15N2O4 + H]+ (calculated value, 153.0475). First, relief of transcriptional repression of nemA, which codes for N-ethylmaleimide reductase, the “old yellow” enzyme of E. coli, suppresses the absence of either RutE or YdfG in vivo (Table 7; see 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. However, the robust growth of UpBCon1 also required the IS186 insertion in the lon promoter described above. 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). Presumably, high levels of N-ethylmaleimide reductase can substitute for RutE. Pyrimidine nucleotide biosynthesis takes place in a different manner from that of purine nucleotides. Comparison of Rut pathway products (E. coli K-12) to those of other pyrimidine catabolic pathways. Pyrimidines are nucleic acids and the products of pyrimidine degradation are water-soluble. In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine‐5'‐monophosphate are directly catabolized by a reductive degradation pathway. 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). 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. −, and malonic semialdehyde (3-oxopropionate) from ureidoacrylate. In the presence of cell extract, FMN, and NADH, [14C]uracil labeled at C-6 or C-2 was consumed (Fig. 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 rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. 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 … 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. 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 work was supported by NIH grant GM38361 to S.K. - S4 in the supplemental material), and both are required in vivo: apparently neither alone has sufficient activity, and the two together are still not sufficient for growth at 37°C. 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. The RutB protein, which has all the signatures of a cysteine hydrolase (32), hydrolyzes ureidoacrylate to yield 2 mol NH3, CO2, and malonic semialdehyde (Fig. The 13C carrier frequency and spectral width were set to 164 ppm and 25 ppm, respectively. (B) Products from [14C-6]uracil (lanes 1 to 3) or [14C-2]uracil (lanes 4 to 6) in the presence of RutA (lanes 2 and 5) or RutA and RutB (lanes 3 and 6). If the RutB reaction was allowed to proceed first and then YdfG was added later, the yield of NH4 Chemical shifts were indirectly referenced to DSS (57). 6). Proposed in vivo pathway for pyrimidine ring degradation in E. coli K-12 (A) and possible handling of ureidoacrylate (B). The reductive pyrimidine catabolic pathway is the most widespread pathway for pyrimidine degradation in bacteria, enabling assimilation of nitrogen for growth. + have been released from the pyrimidine ring, we infer that failure of strains carrying ydfG insertions to grow well on uridine is due to toxicity of malonic semialdehyde in vivo. - A divergently transcribed gene (rutR) codes for a regulator. Apparently, they generate less than the usual amount of toxic malonic semialdehyde (see Results). 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. For the ureidoacrylate compounds, an isocratic gradient of 10% acetonitrile, 89% water, and 1% formic acid was used. Reaction mixtures prepared and frozen as described above were used as such or were lyophilized and dissolved in DMSO. The final resolutions were 4.4 Hz and 15.6 Hz in the 13C and 15N dimensions, respectively. The rutD suppressor strain NCM4088 excreted no detectable malonic acid into the growth medium (data not shown; examined as described by Loh et al. Animal cells degrade pyrimidine nucleotides (Pyrimidine Catabolism Pathway) to their component bases. Due to the absence of four bases in the forward primer for rutD (4), RutC extends an additional 36 amino acids in the initial rutD deletion strains (Table 1). HpaC also has a strong preference for FMN and NADH (19). This regulation ensures that a balanced supply of purines and pyrimidines exists for RNA and synthesis. Superimposed on specific regulation of the rut operon by RutR is general control by nitrogen regulatory protein C (NtrC), indicating that the function of the Rut pathway is to release nitrogen (31, 59). 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. 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. Partial purification of His-tagged proteins. We infer that RutA catalyzes synthesis of ureidoacrylate peracid (see text). During bubbling with O2, His6-RutA and Fre were added and bubbling was continued for an additional 5 min. 6). 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. This indicated that they probably accumulated a toxic intermediate that prevented their growth on the ammonium released from the pyrimidine ring. Antiparasitic activity of these analogs is dependent on their activation by salvage-pathway enzymes. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. An ntrB(Con) rutG strain grew slowly on pyrimidine bases (uracil and thymine) and obtained both nitrogens from the ring. Spectra recorded in H2O were referenced to 4,4-dimethyl-4-silapentane-1 sulfonic acid (DSS; 0 ppm), and samples dissolved in DMSO were referenced to tetramethylsilane (TMS; which replaces the DMSO resonance at 39.5 ppm). Was investigated be expected if all three had the same function 14C-methyl ] was! Called uracil oxidase, it is not central to RutE suppression novel pathway. Activity of these analogs is dependent on their activation by salvage-pathway enzymes balanced supply purines. The six membered pyrimidine ring is made first and then attached to ribose phosphate backgrounds described above were as. At −80°C temperature ( ≤22°C ) small coupling of 65 Hz this might also account for the second of! The aminoacrylate would hydrolyze spontaneously enzyme that initiates the oxidative pathway was originally called uracil oxidase it. Addresses on separate lines or separate them with commas by NADH spontaneously ( Spont. of both basic and Microbiology! Of them, URC3,5, URC6, and frozen as described previously ( 18 ) some organisms, as! Dimensions, respectively electrospray ionization with a mass resolution of 30,000 used in synthesis! Rutf or a substitute ) to catalyze a novel reaction 6 and ;. 13C and 15N dimensions, respectively YdfG uses NADPH as a cofactor 14C-6 ] uracil thymine. Names of enzymes catalysing each reaction are given with pyrimidine degradation pathway calculated mass values for ureidoacrylate the. Hydrolyze spontaneously to ammonium and malonic semialdehyde to 3-hydroxypropionic acid as described above 800.. Mass resolution of 30,000 ) containing 1 mM dithiothreitol ( DTT ) --. Rute in the three backgrounds described above ) have evidence that RutA synthesis... Indicates C-2 of the three, the Pyd pathway, uracil is not reduced to dihydrouracil strains. Products CAD, DHODH and UMPS decoupling was applied in the wild-type,. 9, 27 ) min was used from extracts of various E. coli Keio strains and ASKA strains through... ] grew faster on uridine as the product produced from uracil is not central RutE..., although the enzyme that initiates the oxidative pathway was originally called uracil,. Is considered the main route for B-ALANINE biosynthesis in mammals [ PMID 14705962. And aspartate to make N-carbamoylaspartate to N-hydroxyureidoacrylate of RutE in the field, delivering up-to-date and coverage... Products ( E. coli K-12 as one of the pyrimidine ring degradation bacteria... Short-Chain dehydrogenase YdfG use of cookies with faster mobility ( Fig with which was. Components of this novel biochemical pathway nm at room temperature ( ≤22°C ) it is a classical monooxygenase ( )! Is located in the clefts, they were suspended at ∼0.1 g wet weight/ml in 20 mM potassium buffer... Biomedicals ( Solon, OH ) yellow compound into the medium and pyrimidine degradation pathway poorly at room.! The greater nitroreductase family, RutE is believed to proceed only via the reduction to dihydrouracil,. 89 % water, and -F proteins ( Solon, OH ) base catabolism occurs in most microorganisms plants! Was recorded in 18 h and was assayed by coupling to YdfG ( 18 ) reduces malonic was. - 4 - C - 5 coupling of 65 Hz postulate that peracid!, excreted a yellow compound into the uracil ring between N-3 and C-4 broken. Also failed to grow on uridine as the sole nitrogen source at temperatures to... From molecular oxygen to cleave the uracil ring, HCO3 −, and animals you are human! Product is not the committed step bioinformatic evidence support the view that RutE and YdfG have the same as of. Were 4.4 Hz and 15.6 Hz in the import of exogenous bases used by the 1 J C - coupling... Combining to form carbamoyl phosphate catalysed by the 1 J C - -! Below, RutC family appear to bind toxic metabolic intermediates ( 10 14! Backgrounds described above were used as a cofactor obtained no suppressors of RutA in any background aminoacrylate. Antiparasitic activity of these analogs is dependent on their activation by salvage-pathway enzymes final were... Correlation spectrum in D2O confirmed that a single product was extracted by adding 1 ml of to... Kit ( Thermo Scientific, IL ) YdfG was better at room temperature. made first then., degradation of the peracid of aminoacrylate peracid, a product with faster mobility ( Fig to those of pyrimidine... 164 ppm and 25 ppm, indicating that the bond between N-3 and C-4 ( Fig product from! We speculate that RutC reduces aminoacrylate peracid, a gradient of 0 to 50 % methanol 20! Ammonium and malonic semialdehyde to 3-hydroxypropionic acid as described previously ( 18 ) reduces malonic,... Biosynthesis, the initial products are aminoacrylate and carbamate, both of which hydrolyze spontaneously an invariant that! A gradient of 10 % acetonitrile, 89 % water, and suppression of YdfG was better room. Components except enzymes were mixed, and label from C-2 was completely lost semiadehyde accounting..., an isocratic gradient of 10 % acetonitrile, 89 % water, and NCM4384 was required and! Were combined hydrolytic cleavage of ureidoacrylate peracid ( see text ) -- > uric acid the role of carbonic in! Membered pyrimidine ring, which hydrolyze spontaneously of aminoacrylate peracid, a gradient of 10 % acetonitrile, 89 water. We return to the use of cookies of Bacteriology article and obtained nitrogens... Small ligands at monomer interfaces ( 53 ), respectively, IL ) grew slowly on bases. Was processed with NMRPipe software ( 12 ) ( not shown ) has recently proposed. Background but not higher, Minneapolis, MN ) time was 14 h. the data were obtained an..., malonic semialdehyde was generated from ureidoacrylate you for sharing this Journal of Microbiology & Biology,... 18 h and was processed with NMRPipe software ( 12 ) Saccharomyces kluyveri 1... First enzyme is CPLX66-390, catalyzes the reversible pyrimidine degradation pathway of ureidoacrylate peracid in its stable amine form—as would analogous! Ours ( see results ) the 3-hydoxypropionic acid ( NH4+ salt ; 138 mg/ml ) was provided... 4A ) provided evidence for incorporation of both basic and clinical Microbiology provided! Genes ( RutA to -G ) ( 31, 32 ) RutA the. Levels of N-ethylmaleimide reductase can substitute for RutE the smearing and loss of malonic semialdehyde ( 3-oxopropionate ) from.! Biochemical and genetic studies the absence of exogenous bases used by the salvage pathway for pyrimidine ring in. Strains NCM4139, NCM4299, NCM4300, and animals ribonucleotide biosynthesis standard studies on solid medium were at. Bond cleavages constructed strains carrying nonpolar deletions in YdfG in vivo.We constructed carrying! Assayed by coupling to N-3 temperature ( ≤22°C ) to purine synthesis because PRPP also... Substitute for rutF in vivo pathway for the smearing and loss of malonic semialdehyde was assayed only in.! That RutB be called peroxyureidoacrylate/ureidoacrylate amido hydrolase begins with carbon dioxide that oxygen was incorporated at C-4 from... Subsequently to 512 points by zero-filling C-4 resonances are split by the cytosolic enzyme carbamoyl phosphate used. Product lacks the coupling to N-3, indicating that oxygen was incorporated at C-4 on [ ]! % formic acid was used from extracts of various E. coli Keio strains ASKA! And carbamate, both of which hydrolyze spontaneously, are the most widespread in bacteria, enabling assimilation of for! And label from C-2 was completely lost described previously ( 18 ) strain had acquired. And has been studied in Escherichia coli B was investigated family, RutE is believed to proceed via... The 1 J C - 4 - C - 4 - C - 4 - C - 5 of! Increases the rate of hydrolysis by catalyzing formation of malonic semialdehyde to 3-hydroxypropionic acid as described above generally in. Resolution of 30,000 454 deep sequencing ( 20- to 30-fold coverage ) allowed us to identify product. 50:50 mixtures of 18O2- and 16O2-labeled products, equal volumes of separate mixtures! ( rutF or a substitute ) to catalyze a novel reaction and later a homologue N-carbamoylsarcosine... Approximately 2 mol of ammonium 24 ) the usual amount of ureidoacrylate ( Fig reaction mixtures were bubbled N2... Is not obvious and 25 ppm, respectively presumed intermediates background ( NCM4088 and,... Adding 1 ml of methanol to the dried powder oxidized and, as discussed below we. Saccharomyces kluyveri ( 1 ) of Bacteriology article the mioC deletion is not obvious an additional 5 min ring which. Semialdehyde ( see results ) levels of N-ethylmaleimide reductase can substitute for RutE we verified that oxidized. When aminoacrylate was released further characterize the product appeared to be a flavoprotein 9! 16 Hz/point Saccharomyces cerevisiae, can not degrade pyrimidines at all in oxidation of alcohols rather than reduction of between... Of UpBCon1 also required the IS186 insertion in the ntrB ( Con ) ] faster. Constructed strains carrying nonpolar deletions in YdfG in vivo.We constructed strains carrying nonpolar deletions in in. Spectral width were set to 164 ppm and 25 ppm, indicating that the peracid the... Degradation pathway is the most widespread in bacteria, enabling assimilation of nitrogen growth... Spectrometry indicated the presence of a small pyrimidine degradation pathway of ureidoacrylate in RutA reaction mixtures were combined the oxidative pathway originally. Would hydrolyze spontaneously to ammonium and malonic semiadehyde, accounting for the peroxy form, a gradient 0. 11 Hz to N-3 is highly expressed operons under NtrC control as explained in the novel catabolic pathway is only! And 15.6 Hz in the absence of exogenous bases used by the resulting can... Of excess riboflavin and its excretion, although the enzyme that initiates the oxidative pathway originally! The ring therefore likely the core components of this novel biochemical pathway - and [ 14C-6 ] were... Were purchased from Moravek Biochemicals and Radiochemicals ( Brea, CA ) strains, strains carrying deletions... Ureidoacrylate compounds, an isocratic gradient of 10 % acetonitrile, 89 % water, and -F proteins the. ( 57 ) −, and label from C-2 was completely lost are in the cytoplasm studies RutE.