Rut Pathway for the Degradation of Uracil

Before 2006, uracil and thymine catabolism were thought to occur by two well-known pathways, either oxidative or reductive.  In 2006, Kustu and co-workers (2) described a new pathway for pyrimidine degradation in E. coli, which is required for growth from uracil as the sole nitrogen source. The system known as the rut operon consists of seven previously uncharacterized proteins which are involved in the conversion of uracil to ammonia, carbon dioxide,and 3-hydroxypropionic acid.  We used mass spectroscopy and NMR spectroscopy to characterize the product of the first enzyme in the pathway, Rut A, using uracil as the starting compound (3).



Uracil is cleaved between N3 and C4


1D 13C spectra of uracil (right) and reaction product (left) at 600 MHz.  The reaction was conducted with 15N/13C labeled uracil.  In uracil, C4 is coupled to C5 (65 Hz) and to N3 (11 Hz).  For the product, the coupling between C4 and N3 is lost, indicating that the bond between N3 and C4 has been cleaved.  The different chemical shift of the species in A and B result from the fact that the spectrum of uracil was recorded in H2O, while spectrum of the product was recorded in DMSO.


N3 is converted into a NH2 group


Carbon-detected 13C-15N HSQC spectrum of the Rut A reaction product starting from uniformly 15N/13C - labeled uracil at 900 MHz.  As expected, N1 correlates with both C2 and C6, whereas N3 correlates only with C2.  The experiment was recorded without 1H decoupling in the 15N dimension. As a result, the N3 resonance is split into a triplet, (1JNH ~ 90 Hz inset), indicating that N3 is a NH2 group. The spectrum was recorded at 900 MHz with a carbon-optimized CP-TCI cryoprobe.



Evidence that enzyme Rut A incorporates an oxygen atom from O2 at position C4


1D 13C spectra of the C4 position of uracil (left) and reaction product (right) at 600 MHz. Two reactions were started using uracil labeled with 13C at positions C4 and C5.  In one reaction, 16O2 was bubbled into the solution, and in the second 18O2 was bubbled in. For uracil, a one-bond coupling between C4 and C5 of 65 Hz is observed. For the reaction product, in addition to the C4-C5 coupling, an 18O shift is observed (arrows), indicating that an an oxygen atom from molecular oxygen is incorporated at position C4.

References:

  1. Loh, KD, Gvaneshwar, P, Markenscoff, PE, Fong, R, Kim, KS, Parales, R, Zhou, Z, Inwood, W, Kustu, S. Proc. Natl. Acad. Sci. 2006, 103, 5114-5119.
  2. Kim, KS, Pelton, JG, Inwood, WB, Andersen, U, Kustu, S, Wemmer, DE, J. Bacteriol, 2010, 192, 4089-4102.