Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium
Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium
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DOI：
10.1016/j.jmb.2004.01.039
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发表时间：
2004-03-19
影响因子：
5.6
通讯作者：
Cutfield, JF
中科院分区：
文献类型：
--
作者：
Caradoc-Davies, TT;Cutfield, SM;Cutfield, JF
尿苷磷酸化酶（UP）是嘧啶补救途径中的关键酶，催化尿苷可逆磷酸化为尿嘧啶和核糖I-磷酸。抑制人类肝脏UP可提高血液尿苷水平，并产生针对化疗剂5-氟尿嘧啶毒性的保护作用（“尿苷拯救”），而不会降低其抗肿瘤活性。我们研究了UP-底物相互作用，通过确定天然大肠杆菌UP（两种形式）的晶体结构，并与5-氟尿嘧啶/核糖I-磷酸，2-脱氧尿苷/磷酸和胸苷/磷酸复合物。这些六聚体结构证实了UP与E的总体结构相似性。大肠杆菌嘌呤核苷磷酸化酶（PNP），其中，在底物存在下，每一个都显示出闭合构象，这是由闭合活性位点裂缝的协同运动产生的。然而，与螺旋分段是开放和闭合形式之间的主要构象变化的PNP相反，在UP中观察到更广泛的变化。特别地，由残基224-234组成的翼片区域的摆动运动密封活性部位。这种构象的整体变化导致活性位点裂缝的压缩。Gln 166和Arg 168是PNP中未见的插入片段的一部分，是尿嘧啶结合口袋中的关键残基，与紧密结合的水分子一起参与UP的底物特异性。酶活性显示出对钾离子浓度的双重依赖性。钾离子在单体/单体界面处的存在诱导一些局部重排，这导致二聚体稳定化。磷酸和核糖结合口袋中关键残基的保守性以及与底物的相互作用表明，UP催化过程中的核糖氧碳正离子形成可能与E. coli PNP。(C)2004爱思唯尔有限公司保留所有权利。
Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose I-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose I-phosphate, 2-deoxyuridine/ phosphate and thymidine /phosphate. These hexameric structures confirm the overall structural similarity of UP to E. coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E. coli PNP. (C) 2004 Elsevier Ltd. All rights reserved.