The uracil and product C-4 resonances are split by the 1 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. Based on the results presented above, the mioC deletion is not central to rutE suppression. Apparently, they generate less than the usual amount of toxic malonic semialdehyde (see Results). S4 in the supplemental material). We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. Requirement for YdfG in vivo.We constructed strains carrying nonpolar deletions in ydfG in the three backgrounds described above. This indicated that the uracil ring had been cleaved between N-3 and C-4 (Fig. 2A and B) (data for other strains not shown): rather, as radiolabeled uracil (C-2 or C-6) was consumed, the products of the RutB reaction appeared, indicating that hydrolysis by RutB was much faster than the RutA/F reaction. The synthesis begins with carbon dioxide and ammonia combining to form carbamoyl phosphate catalysed by the cytosolic enzyme carbamoyl phosphate synthetase-II. Xanthine Oxidase. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. 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. 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. 2A): at most, traces of RutA/F product were observed. The RutF protein was not well overexpressed and was assayed only in extracts. Presumably, high levels of N-ethylmaleimide reductase can substitute for RutE. Based on biochemical evidence, RutC was originally predicted to be an endoribonuclease (31, 36), but recently this has been questioned (32; see Discussion). Protein concentrations were determined using the Micro bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, IL). All components except enzymes were mixed, and reaction mixtures were bubbled with N2 for 5 min. Cells were harvested and frozen at −80°C until use. 5). 5B). 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 question is for testing whether or not you are a human visitor and to prevent automated spam submissions. S2B in the supplemental material). 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. 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). The presence of the correct product was confirmed by comparing 1H-nuclear magnetic resonance (NMR) chemical shifts and coupling constants in dimethyl sulfoxide (DMSO) with published values (16). beta-Ureidopropionase deficiency is an inborn error of the pyrimidine degradation pathway, affecting the cleavage of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid. In the presence of cell extract, FMN, and NADH, [14C]uracil labeled at C-6 or C-2 was consumed (Fig. We postulate that they act in order on the 3-carbon intermediate released by RutB (Fig. Evidence that RutE and YdfG have the same function. 6). Residual growth may be accounted for by the fact that the ntrB(Con) lesion activates transcription of the gene(s) for other transporter(s) that can carry pyrimidine nucleosides/bases (59). 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. 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). When aminoacrylate was released by RutC, RutD could then increase its spontaneous rate of hydrolysis by catalyzing formation of the rare imine tautomer. For the peroxy form, a gradient of 0 to 50% methanol over 20 min was used. (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). Finally, overexpression of N-ethylmaleimide reductase allows an ntrB(Con) strain, which expresses the rut operon at high levels, to grow on pyrimidines as the sole nitrogen source at 37°C (although not as well as the UpBCon2 strain; see Results). To see whether RutB would hydrolyze ureidoacrylate, the product we obtained from the RutA/F reaction, we first used RutA and the substitute flavin reductase Fre to prepare radiolabeled ureidoacrylate from uracil. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. Comparison of Rut pathway products (E. coli K-12) to those of other pyrimidine catabolic pathways. They were then bubbled with 18O2 or 16O2 for 1 min. 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). ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology. Names of enzymes catalysing each reaction are given with the AGI locus and gene name. In plants, the pyrimidine bases, uracil, and thymine, derived from uridine monophosphate and deoxythymidine‐5'‐monophosphate are directly catabolized by a reductive degradation pathway. Synthesis of Z-3-ureido-2-propenoic acid. The rutF deletion extended an additional 12 bp beyond its predicted C-terminal end but remained in-frame and within rutF. Materials for the RutA/F reaction. Apparently no other flavin reductase can substitute for RutF in vivo. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. under conditions similar to ours (see Discussion and Fig. We verified that YdfG oxidized serine and 3-hydroxypropionic acid as described previously (18). - + was released. 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). 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. 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. 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. 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). Although the enzyme that initiates the oxidative pathway was originally called uracil oxidase, it is a classical monooxygenase (28). The rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. (A) RutA/F reaction. 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. In the clefts, they carry an invariant R that is often followed by XC. 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. 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. Samples in lanes 1 and 4 are from controls with no enzyme. 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. Necessary to delete it unless kanamycin sensitivity was required spectra were taken in DMSO 50 % methanol over 20 was! Mukherjee and Tadhg Begley, Cornell University and Texas a and M University, respectively RutC activity: at a... One mole of ammonium, malonic semialdehyde to 3-hydropropionate obtained mass spectral data on the presented... 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