The enzyme Phosphoribosyl pyrophosphate (PRPP) synthetase (PDB ID: 3LRT) catalyzes the transfer of a pyrophosphate group from ATP to ribose-5-phosphate (R5P), yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in many life processes, such as cellular metabolism. PRPP is involved in the biosynthesis of purine and pyrimidine nucleotides, coenzyme NAD+ and the amino acids histidine and tryptophan; it is also a substrate for many enzymes (1). 

PRPP synthetase from the thermophilic archaeon, Thermoplasma volcanium (Tv), is an asymmetric unit containing subunits A and B that form a biological homodimer. Each subunit contains an N-terminal and C-terminal domain. Each domain contains an open α/β fold, a central six-stranded parallel β-sheet and four helices that surround the β-sheet. The N-terminal domain is surrounded by an extra helix from the C-terminal domain. β-hairpins in both domains play a role in the enzymatic function of PRPP synthetases. The β-hairpin of the N-terminal domain forms part of the active site to adjoin the dimer. The C-terminal β-hairpin loop is catalytic and can adopt two conformations, open and closed (1). 

Tv PRPP synthetase comprises binding sites for ATP and R5P. The ATP and R5P binding sites are located along the β-10 strand along with catalytic residues Lys-184 and Arg-186; these binding sites and residues comprise the active site of Tv PRPP synthetase. There are two independent active sites in the Tv PRPP synthetase dimer, each in a cleft between the two domains of each subunit.  Tv PRPP synthetase adopts an open and closed conformation throughout the stages of its catalytic cycle, affecting the active sites. When each subunit has an open conformation, the active sites are accessible for ATP and R5P to bind. Catalysis happens during the closed conformation (1).

Hydrogen bonds and electrostatic interactions play a role in the binding of substrates ATP and R5P to Tv PRPP synthetase, namely the network of hydrogen bonds and electrostatic interactions between Tv PRPP synthetase and ADP. Tyr-96 and His-93 electrostatically interact with the β-phosphate of ADP and Asp-34 and Glu-36 form hydrogen bonds with the nitrogen atoms of the adenine ring (1).

ADP, the byproduct of the transfer of the pyrophosphate group from ATP to R5P, is a ligand present in Tv PRPP synthetase. Another ligand present is a sulfate ion that occupies the R5P binding site. The sulfate ion originates from the crystallization liquid used to crystalize the structure of Tv PRPP synthetase. The sulfate ion mimics the phosphate group of R5P. It forms hydrogen bonds to two residues in the phosphate-binding segment of the binding site. The hydrogen bonds are mainly between main-chain nitrogen atoms and to hydroxyl groups of threonine residues. It also forms a salt bridge with the guanidinium group of Arg-91 (1). 

Bioinformatics searches found proteins with similar primary or tertiary structures to Tv PRPP synthetase. A PSI-blast found subjects, or proteins with similar nucleotide sequences to the protein query, Tv PRPP synthetase. A PSI-blast assigns an “E value” to subjects that have sequence homology to the query. The E value expresses the total sequence homology by assigning gaps, or amino acids that exist in the subject’s sequence but not the query. The higher the E value, the more gaps. An E value of less than 0.5 is significant for proteins. The Dali Server compared the tertiary structures of other proteins to Tv PRPP synthetase. It uses a sum-of-pairs method to compare intramolecular distances, providing a measure of similarity measured by Dali-Z scores. Tertiary structures with significant similarity to Tv PRPP synthetase have Z-scores greater than two. These similar structures usually have similar folds (2,3).  

Tv PRPP synthetase has a significant sequence homology to PRPP synthase of the hyperthermophilic autotrophic methanogenic archaeon, Methanocaldococcus jannaschii (Mj), with a Dali-Z score of 31, and the low PSI-blast E value of 8e-36 (2,3). Mj PRPP synthase (PDB ID: 1u9y) is another class of PRPP synthetase. It is activated by phosphate and magnesium ions, and is independent of allosteric regulation. Opposed to the dimer, Tv PRPP synthetase, Mj PRPP synthase forms a tetramer with two dimers. Superposition of a Tv dimer on an Mj dimer shows major differences between the two. The β-hairpin of Tv PRPP synthetase (residues 179-187) is in a different orientation compared to the β-hairpin of the Mj PRPP synthase (residues 185-196) (1,6). The respective molecular weights and isoelectric points of Tv PRPP synthetase and Mj PRPP synthase are: 32,177.46 Da and 5.66, and 31,395 Da and 5.39 for the latter (4).

Class I synthases occur in a range of bacteria, Class II synthases are found in plants and Class III is represented by the enzyme from Mj. The structure of Tv PRPP synthetase is similar to Class I and Class III synthases. Tv PRPP synthase also has the highest sequence homology to Mj PRPP synthetase, with a sequence identity of 29%. Similar to Tv PRPP synthetase, the Mj synthase has identical phosphate-binding residues to the residues in Tv PRPP synthetases R5P binding site (1,5).

After comparing the two, it is found that the function of Tv PRPP synthetase and MJ PRPP synthetase are the same. However, the differences in structure have possible implications of the catalytic mechanisms of each (1).