PRibosylPPSynthetase1

Phosphoribosylpyrophosphate (PRPP) synthetase 1 (PDB: 2H06) from Homo sapiens (hPRS1) belongs to the PRPP synthetase (PRS) class of enzymes that catalyze the synthesis of PRPP from ATP and ribose 5-phosphate (R5P) by transferring the β,γ-diphosphoryl moiety of ATP to the C1-hydroxy group of R5P (1). PRPP serves as an important intermediate for creating purine and pyrimidine nucleotides, NAD and NADP, and the amino acids, histidine and tryptophan. All of these products from the PRPP intermediate are essential to life as coenzymes, amino acids in proteins, and nucleic acids in DNA, among other roles. Because PRS form this important intermediate, PRSs are found in a variety of organisms with similar structures; human genes encode for three similar isoforms of PRS, of which hPRS1 is the most studied (2). The molecular weight of hPRS1 is 71,780.69 Da, and its isoelectric point (pI) is 6.58 (3).

hPRS1 consists of two subunits that form a homodimer and interacts with six sulfate ligands. Subunit A contains 305 amino acid residues; whereas subunit B has 308 residues, but they are essentially identical as revealed by superposition of Cα atoms. Each subunit has two domains that form an α/β “sandwich” structure with a variety of secondary structures. The N-terminal domain has a central five-stranded parallel β sheet surrounded by four α-helices and one 3₁₀-helix, and the C-terminal domain is flanked by two α-helices on one side and three α-helices on the other (1). Three of these hPRS1 homodimers form a hexamer in a propeller shape with 32 point group symmetry.

The interface of two domains of one subunit contains the active site for ATP and R5P. The active site is composed of a flexible loop formed by residues Phe-92—Ser-108; the pyrophosphate binding loop of residues Asp-171—Gly-174; and the flag region of an adjacent subunit, residues Val-30—Ile-44 (4). ATP typically interacts with an Mg²⁺ ion or another divalent cation as shown by biochemical data before it interacts as the substrate to the active site, allowing for the enzyme to cleave pyrophosphate from ATP and forming AMP (5). The ATP interacts with a Mg²⁺ cation or other cation in order to help with stabilization of the ATP within the enzyme resulting in a Mg-ATP complex, which acts as the substrate for the enzyme. Once formed, AMP has several hydrogen bonds with Arg-96, Gln-97, Asp-101 of subunit A and Asn-37 and Glu-39 of subunit B using its adenine N1 and N6 atoms and its ribose 2’-and 3’-hydroxy groups. Its α-phosphate has hydrogen bonding with His-130 and Lys-99. Arg-196 is closely located to the α-phosphate of ATP suggesting that it interacts with the pyrophosphate during enzymatic activity and helps to stabilize the transition state. A bound Cd²⁺ ion, which can mimic the Mg²⁺ in the complex and was used in place of Mg²⁺ for determining the crystal structure, interacts with the Asp-171 and Asp-220 along with interacting with β- and γ- phosphates of ATP to better stabilize the ATP molecule in the active site . The R5P binding loop is located between Asp-220 and Thr-228 and contains a sulfate ion that can directly hydrogen bond with the main chain amides of Asp-224, Thr-225, Cys-226, Gly-226, and Thr-228, as well as the side chains of the mentioned threonine residues (4). Arg-96 is possibly involved in both the binding of ATP and the binding and release of PRPP because it has been shown to have two different conformations in the two different subunits. One has a conformation with the Asp-224 of the R5P binding loop and the other conformation points towards the side chain of Asp-171 of the pyrophosphate binding loop (4). The side chain of Arg-9, however, has the same conformation for both subunits when ATP is present in the binding site allowing it to form hydrogen bonds with the ATP and the binding loop. Arg-96, therefore, appears important to ensuring the functionality of the enzyme.

ADP is the most important inhibitor because it can bind at both the ATP binding site and competitively bind with phosphate at the allosteric site. ADP and phosphate compete for binding at a common allosteric site that consists of conserved residues Gln-135, Asp-143, Asn-144, and Ser-308 to Phe-313 of one subunit, residues Lys-100—Arg-104 of the flexible loop of the other subunit, and residues Ser-47, Arg-49, Ala-89, and Ser-81 of the third subunit. Sulfate can also mimic the properties of phosphate for activating the enzyme and forms hydrogen bonds with the side chains of Ser-47, Arg-49, Arg-104, Ser-308, and Ser-310, along with the main chains of Val-109 and Ser-310 (4). Sulfate occupies the position that would otherwise be occupied by the β-phosphate of ADP preventing any inhibition from ADP.

Because PRPP synthetases are common throughout many organisms, there are strong structural similarities between PRSs of different organisms. In particular, hPRS1 shared up to 47% of its primary structure with Phosphoribosylpyrophosphate synthetase (bsPRS) from Bacillus subtilis (PDB: 1DKR) according to comparisons conducted by the PSI-BLAST program, which assigns an E-value based on similarities in protein sequences. The value increases when there are gaps in the sequence of the compared protein, so an E value of 0 indicates a 100% match. The E value for bsPRS compared to hPRS1 is 3E-12, which indicates a very strong match in primary structure of the two proteins considering the cut off is a value of 0.5 (6). Furthermore, the overall tertiary structures of the two proteins can be compared using the Dali server. The Dali server assigns a Z score based on a sum-of-pairs method which measures similarity by comparing intramolecular distances. A Z score greater than 2 indicates that the structures have significant similarities. bsPRS has a Z score of 40.8 when compared to hPRS1 indicating a strong similarity between the two proteins’ tertiary structures (7). A comparison between bsPRS and hPRS1 demonstrates evolutionary conservation of the sequence and tertiary structure of the PRS enzyme from a bacterial cell to a more complex eukaryotic cell of humans (8).

Given the strong similarities between hPRS1 and bsPRS, there remains some variation between the two proteins. An extra allosteric binding site containing a sulfate ion appears to be located in hPRS1 that does not exist in the structure for bsPRS. This unique allosteric binding site is located between the ATP binding site and the previously mentioned allosteric site. The sulfate ion has favorable hydrogen bonding interactions with the side chains of Ser-132, Gln-135, Asn-144, and Tyr-146 of one subunit A along with the main chain amides of Lys-100, Asp-101, and Lys-102 of the flexible loop of subunit B (4). These residues appear to be conserved throughout eukaryotic PRSs such as hPRS1, but can be replaced in bacterial PRSs. The binding of the sulfate ion at this second allosteric site correlates to a substantial conformational change of the flexible loop at the active site. This flexible loop appears partially disordered adopting different conformations in the absence or presence of ATP for bsPRS, whereas it is well-defined in the presence and absence of ATP for hPRS1 (4). The binding site residues were compared with those residues found in the same location in bsPRS and residues modified at mentioned allosteric site in mutants of hPRS1. By conducting enzymatic activity assays, this second allosteric site was found to stabilize the flexible loop important in binding the Mg-ATP substrate (4). This unique binding site within the structure of hPRS1 allows the enzyme to effectively carry out its function and each of the subunits for the binding site appears conserved in most eukaryotic cells. This initial binding is essential to beginning the catalytic reaction and helps prevent the inhibitor ADP from entering the active site. The second allosteric site appears to suggest an evolutionary adaptation from bacterial cells to better regulate the activity of the PRSs due to the higher complexity of eukaryotic cells and is critical to the function of the enzyme within these more complicated organisms.