The crystal structure of Streptococcus pyogenes uridine phosphorylase reveals a distinct subfamily of nucleoside phosphorylases

Abstract

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

Figure 1

Proposed transition state for PNP from human blood (12).

Figure 2

Structures of SpUP. (a) Hexameric quaternary structure of SpUP color-coded to emphasize the 32 point symmetry or a trimer of dimers. Six subunits are labeled A to F. (b) SpUP protomer with the bound products, R1P and Ura, shown in the active site. The helices are colored blue, the strands green, and the loops yellow. The N-terminus and the C-terminus are also indicated with letters N and C. (c) Topology diagram of SpUP protomer. The colors of the helices and strands are the same as Figure 1b, except the loops are depicted as black lines. The first and last residues of each secondary structure are indicated by numbers.

Figure 3

Active site of SpUP. (a) Electron density for uracil and R1P. The NCS-averaged F_o-F_c density was calculated with phases from the refined model after the ligands were removed. The map is contoured at 3.0 σ. (b) Stereo diagram of the active site containing R1P and Ura. The red spheres represent water molecules. Hydrogen bonds are indicated by dashed lines.

Figure 4

Superposition of the active sites of SpUP (green) and EcUP (magenta) in stereo. The spheres are water molecules found in the active site.

Figure 5

The insertion region (gray box) of SpUP is revealed by the alignment of SpUP against EcUP and EcPNP. Magenta, green, and orange triangles indicate conserved residues in the phosphate-binding site, ribose-binding site, and uracil-binding site of SpUP, respectively. The residues marked with an asterisk are the two novel residues found in the uracil-binding site of SpUP.

Figure 6

Active sites of SpUP with uridine and phosphate manually positioned in the active site (a), EcPNP/inosine/PO₄ complex (b), EcMTAN/5′-methylthiotubercidin (c), and EcAMN/formycin 5′-monophosphate (d), respectively (, –33). ‘W’ stands for water. Residues marked with an asterisk come from an adjacent monomer.

Figure 7

Transition state stabilization of high energy intermediates of the phosphorolysis reaction in EcUP (a) and SpUP (b) by a continuum of electrostatic and hydrogen bond interactions as illustrated by the dotted line.