The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Abstract

The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. 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. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of 18 O at C-4 from molecular oxygen indicate otherwise. 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. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. 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. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.The rut (pyrimidine utilization) operon of Escherichia coli K-12 contains seven genes (rutA to -G) (31,38). A divergently transcribed gene (rutR) codes for a regulator. 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). In the presence of uracil, RutR repression of the rut operon is relieved.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). The rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. 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. 1). Waste products are excreted into the medium. (Lactic acid is 2-hydroxypropionic acid.) Wild-type E. coli K-12 can use uridine as the sole nitrogen source at temperatures up to 22°C but not higher. 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).In the known reductive and oxidative pathways for degradation of the pyrimidine ring (22, 48, 52), the C-5-C-6 double bond ...

Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals New URC Genes Involved in Pyrimidine Catabolism

“…We successfully overproduced and purified the encoded protein, Urc8p, and showed that it can catalyze the reduction of a 3-carbon molecule just like its homologs in S. cerevisiae and E. coli, YMR226c and YdfG. The confirmed reaction, i.e., reduction of malonate semialdehyde to 3-hydroxypropionate, is the same reaction catalyzed by RutE and YdfG in E. coli (19). However, the Urc8p homologs YMR226c and YdfG are known multisubstrate enzymes, meaning that Urc8p is likely to be one too.…”

“…These facts together led us to hypothesize that Urc8p performed the same reaction as YdfG. In E. coli, the reaction is proposed to have a detoxifying role, helping to speed up the removal of toxic intermediates formed during the degradation (19).…”

“…Growth was inspected after 3 and 6 days. (23,24), and YdfG is known to reduce malonate semialdehyde to 3-hydroxypropionate in the Rut pathway in E. coli (19), which is also the outcome of the URC pathway (17). We hypothesize that the role of URC8 is similar to that of YdfG.…”

“…To test our hypothesis that Urc8p catalyzes the same reaction as YdfG in the Rut pathway (19), i.e., converts malonate semialdehyde to 3-hydroxypropionate, we used the purified protein in a coupled assay with the ␤-alanine amino-transferase from L. kluyveri, Pyd4p, to make the proposed substrate. ␤-Alanine amino-transferase catalyzes the fourth step of the common reductive pyrimidine degradation pathway, where ␤-alanine is converted to malonate semialdehyde (15).…”

“…In Escherichia coli, 3-hydroxypropionate is pro-duced by the action of RutE or YdfG through the reduction of malonate semialdehyde (19).…”