UMP synthase

Thomas Traut

Research Interests

Dissociating Enzymes

UMP Synthase

Uridine Kinase

Beta-alanine Synthase

Purine & Pyrimidine Concentrations

A. Bifunctional architecture and kinetic properties

With the mammalian UMP synthase, which contains the two catalytic centers orotate phosphoribosyltransferase (OPRTase) and OMP decarboxylase (ODCase), kinetic studies had supported the model shown below. The recent crystal structures of the single domain bacterial OPRTase have demonstrated that each catalytic site is located between the 2 subunits in the dimer. Also, four new crystal structures from 3 bacterial species and one yeast species show that for the single domain OMP decarboxylase the single substrate, OMP, binds at the interface of the 2 subunits. These results help to explain our earlier kinetic studies which had found that only the dimer of UMP synthase had catalytic activity.

As shown in the figure (A), natural gene fusion has cause two genes that are consecutive in microorganisms to become a single larger gene in eukaryotes. Therefore, these two sequential catalytic reactions leading to the synthesis of UMP can be regulated jointly. While this bifunctional enzyme is normally a stable, but inactive monomer, appropriate effectors produce two different and active conformations.



Domain structure of UMP synthase. (A) Two separate genes in microorgnisms become fused in eukaryotes. The single domain proteins in microorganisms are then fused into a single protein with two domains. (B) Sedimentation studies demonstrated how effector ligands, binding at the active site, stabilized the dimeric state. X- repreents a variety of anions; OMP = orotidine monophosphate.


B. What are the benefits of the bifunctional architecture?

Three different and important benefits are possible for the evolution of this architecture:

1. The intact bifunctional protein is more stable in vivo.

2. An element of allosteric control has been achieved.

3. The two catalytic centers may interact, and are thereby individually or jointly more efficient in their catalytic function.

At this time we have strong support for the first two features. The bifunctional protein can be diluted to 10 pM and remain completely stable and fully active, when the concentration of the substrate OMP is = to it's Km. That is, at any moment only half of the protein molecules may be binding with a substrate, while half must be empty.

The two domains, when expressed as distinct proteins, still dimerize with their own subunits, to form fully active enzymes with the sam Km and kcat as the native UMP synthase. But, as these separated domains are diluted before the addition of substrate at a concentration = to its Km, the activity curve becomes quickly nonlinear with time, demonstrating that these proteins are not sufficiently stable, and do not remain dimeric.

Relevant Papers

1. Yablonski, M. J., Pasek, D. A., Han, B.-D., Jones, M. E., & Traut, T. W.  (1996) Intrinsic activity and stability of bifunctional UMP synthase, and of its two separate domains, orotate phosphoribosyl-transferase and orotidine-5'-phosphate decarboxylase.  J. Biol. Chem.271, 10704-10708.   

2. Traut, T.W.  (1989) Uridine-5'-phosphate Synthase: Evidence for substrate cycling involving this bifunctional protein. Arch. Biochem. Biophys. 268, 108-115.  


C. Evolution of OMP decarboxylases

Throughout the evolution of this enzyme:

• Only 8 amino acids (involved in the chemistry or binding of substrate) have remained invariant.

• All other amino acid residues have been mutated in one or more of 82 species known. Such mutations appear not to alter the secondary structure where they occur.

Inother words: all changes are permitted that do not alter the core alpha/beta barrel structure or the chemistry at the active site.

Also, this protein may vary in total size from 202 - 398 amino acids. The data set is large enough to permit some general conclusions:

1. The core structure appears to be strongly conserved, and therefore the core function.

2. Compared to a consensus sequence, random mutations appear in all 82 species, and at all positions except for the invariant 8.

3. Indels (insetions/deletions) occur only in loops.

a) Inserts then add extra amino acids at the surface of the core structure. The picture below shows the position of the most common insertions.

b) It has not been demonstrated that such inserts, even very large ones, add any function to the enzyme.



4. Extra amino acids, above the minimal core, always occur in loops, and such extra polypeptides have not yet been shown to add any additional function to this enzyme.   

Relevant Paper

1.   Traut, T.W. and Temple, B.R.S.  (2000)  The chemistry of the reaction determines the invariant amino acids during the evolution and divergence of orotidine 5'-monophosphate decarboxylase.  J. Biol. Chem. 275, 28675-28681.