Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, United States.
Biochemistry. 2011 Aug 2;50(30):6549-58. doi: 10.1021/bi200707z. Epub 2011 Jul 8.
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.
尿苷磷酸化酶（UP）是嘧啶补救途径中的关键酶，可催化尿苷或 2'-脱氧尿苷的可逆磷酸解，生成尿嘧啶和核糖 1-磷酸或 2'-脱氧核糖 1-磷酸。该酶属于核苷磷酸化酶 I 超家族，其成员对核苷底物表现出不同的特异性。系统发育分析表明，酿脓链球菌尿苷磷酸化酶（SpUP）位于核苷磷酸化酶嘧啶亚家族的一个独特分支中。为了进一步表征 SpUP，我们测定了与产物核糖 1-磷酸和尿嘧啶复合物的晶体结构，分辨率为 1.8Å。与大肠杆菌 UP（EcUP）一样，SpUP 的生物单位是一个六聚体，具有 α/β 单体折叠。活性位点的一个新特征是存在 His169，它与 EcUP 结构中的 Arg168 结构上对齐。第二个活性位点残基 Lys162 不存在于先前确定的 UP 结构中，与尿嘧啶的 O2 相互作用。野生型 SpUP 的生化研究表明，其底物特异性与 EcUP 相似，而 EcUP 的效率比 SpUP 高约 7 倍。SpUP 突变体的生化研究表明，His169 的突变降低了活性，而 Lys162 的突变则完全丧失了活性，这表明过渡态中的负电荷主要位于尿嘧啶 O2 上。这与 EcUP 形成对比，在 EcUP 中，过渡态的稳定主要发生在 O4 上。
