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

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
alpha/beta 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 uracilO2. This is in contrast
to EcUP for which transition state stabilization occurs mostly at O4.