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J Bacteriol. Aug 2010; 192(16): 4086–4088.
Published online Jun 18, 2010. doi:  10.1128/JB.00573-10
PMCID: PMC2916418

The Surprising Rut Pathway: an Unexpected Way To Derive Nitrogen from Pyrimidines[down-pointing small open triangle]

Four years ago, Sydney Kustu's group reported their surprising discovery that Escherichia coli's well-studied genome contains a previously unrecognized seven-gene operon (rutABCDEFG) encoding a pathway to degrade pyrimidines, making the two ring nitrogen atoms available for assimilation (4). The rut operon is one of the most highly expressed clusters of NtrC-controlled genes in E. coli (9), but until the publication of this report, the function of this operon was unknown. Andrei Osterman said he had doubted that such a discovery was possible today: it was “too big a skeleton to be hidden in the closet” (5). The surprises continued as we learned more: the pathway is different from either of the known pathways of pyrimidine degradation (oxidative and reductive) (7): it makes the two ring nitrogen atoms available for assimilation but discards the four ring carbon atoms (one as CO2, the others as a 3-carbon waste product), and it supplies all of the cell's growth-sustaining nitrogen from pyrimidine rings at room temperature but not at 37°C (4).

In this issue of Journal of Bacteriology, the Kustu and Wemmer groups at the University of California, Berkeley, present details about this curious pathway, revealing further surprises (3). A few loose ends remain to be firmed up, but we now know with reasonable confidence what each of the proteins in the pathway does. And it's quite a story. In some ways, rut is a conventional catabolism-encoding operon (Fig. (Fig.11 A). It contains a transporter-encoding gene (rutG) and has an associated, divergently transcribed repressor gene, rutR. But that's where conventionality stops. Three of the Rut proteins and an additional reductase enzyme encoded elsewhere on the chromosome (ydfG) are specifically required to eliminate three highly toxic pathway intermediates. These enzymes are not necessary in vitro—only for the pathway to be tolerated in vivo. And some of the reactions of the pathway, to the best of our knowledge, are unprecedented.

FIG. 1.
The rut gene cluster and proposed pathway for uracil catabolism in E. coli. (A) Genes required for pyrimidine utilization, including rutRABCDEFG and ydfG, which is not colocalized with the rut gene cluster on the E. coli chromosome. Functions of the gene ...

The first enzyme in the pathway (encoded by rutA), which the authors call “pyrimidine oxygenase,” sets the style. In what the authors term the “RutA/F reaction,” the pyrimidine ring is cleaved directly (without prior modification of its aromaticity as occurs in other pyrimidine-degrading pathways) (7), by adding a pair of oxygen atoms and producing, most probably, a peracid (ureidoacrylate peracid; Fig. Fig.1B).1B). This odd metabolite, a strong and highly toxic oxidizing agent, is reduced spontaneously, albeit slowly, to ureidoacrylate. RutF functions as a flavin reductase that is required to regenerate the flavin mononucleotide (FMN) cofactor.

The second enzyme of the pathway (RutB) is an amidohydrolase that cleaves ureidoacrylate in vitro and most likely cleaves the peracid in vivo. One of the products of hydrolysis, carbamate, hydrolyzes spontaneously, thereby releasing one of the pyrimidine ring's nitrogen atoms as ammonia and one of its carbons as CO2. The other product also hydrolyzes spontaneously, releasing the second ring nitrogen as ammonia (Fig. (Fig.1B1B).

At this point, the pathway has completed its purpose: the two pyrimidine ring nitrogen atoms have been released. However, there is an in vivo requirement for two additional Rut proteins, RutC (a reductase) and RutD (a hydrolase), apparently because certain reactions like the spontaneous reduction of ureidoacrylate peracid occur too slowly and additional toxic side products are formed. Ultimately, the resulting malonic semialdehyde presents yet another metabolic challenge: the pathway cannot function in vivo unless the malonic semialdehyde is reduced because it too is apparently toxic. (It can form adducts to free amino groups.) So two other enzymes, RutE and YdfG (2), are needed to convert malonic semialdehyde to 3-hydroxypropionate (Fig. (Fig.1B).1B). This alcohol, which is an active metabolite in many bacteria, is excreted unused by E. coli.

The Rut pathway is as enigmatic as it is unusual: it degrades exogenous pyrimidines as the sole nitrogen source at room temperature but not at 37°C, a restriction that is apparently a consequence of an inadequate ability to remove toxic malonic semialdehyde at the higher temperature (RutE/YdfG function). Consequently, previous large-scale screens apparently missed identifying pyrimidines as nitrogen sources since growth was tested at 35°C (1, 8). It is a catabolic pathway that is highly expressed even in the absence of exogenous substrate, and it is expressed at 37°C (9). The pathway also wastes the carbon atoms of its substrate, excreting them as 3-hydroxypropionate, a compound readily utilized by other microbes.

With these enigmas in mind, it is hard to imagine how this pathway evolved as a means of acquiring nitrogen from pyrimidines. Presumably all of the detoxifying enzymes must have been present before the reactions that generate the toxic intermediates were acquired; thus, the detoxifying enzymes must have had alternative “native” functions. The true role of the pathway is also a mystery. The cluster encodes a transporter, suggesting that at least under some conditions, uptake and utilization of exogenous pyrimidines occur. The authors, however, suggest that the pathway might function to recover nitrogen in endogenously generated pyrimidines from turned-over RNA, which introduces another dilemma. We note that nucleotides so generated can be reused with greater metabolic benefit via E. coli's well-established pyrimidine salvage pathways. The authors also offer the intriguing suggestion that perhaps the pathway's toxic intermediates may also contribute to metabolism and that, under nitrogen-limiting conditions, they may help slow growth in a coordinated manner. We wonder if the pathway might be particularly valuable following a massive nutritional and temperature shift-down, when synthesis of stable RNA ceases as its degradation begins, and mRNA synthesis declines as its turnover continues. Under such conditions, which are imposed when E. coli leaves the mammalian gut to enter the external environment, it would be more metabolically advantageous to degrade internally generated nucleotides than to salvage them. This function for the pathway would be consistent with its higher activity at lower temperatures.

The rut gene cluster is present in many Proteobacteria, with quite a few variations. For example, in some cases, a transporter is not encoded in the cluster, suggesting the possibility that in certain organisms the Rut pathway might function solely to recycle pyrimidines from RNA turnover; however, the presence of alternative pyrimidine transporters encoded elsewhere in these genomes cannot be ruled out. Many of the Rut pathway-containing Proteobacteria are soil organisms, so this pathway is not only associated with a lifestyle shift from host to environment. We suspect that studies on the expression of the Rut pathway in other bacteria with different lifestyles will shed light on the pathway's physiological function(s). It will be instructive to learn, for example, if the Rut pathway is expressed temperature conditionally and if 3-hydroxypropionate is excreted or utilized in other microbes.

Acknowledgments

We thank Michele Igo and Jack Meeks for helpful suggestions and Juan Parales for generating the figure.

Notes

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 June 2010.

REFERENCES

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