Finer-Moore J S, Maley G F, Maley F, Montfort W R, Stroud R M
Department of Biochemistry and Biophysics, University of California at San Francisco 94143-0448.
Biochemistry. 1994 Dec 27;33(51):15459-68. doi: 10.1021/bi00255a028.
Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.
噬菌体T4的胸苷酸合成酶（T4TS）是受感染的大肠杆菌细胞中协调DNA合成所需的几种酶组成的复合物的一部分。有人提出，与DNA合成相关的类似酶复合物在真核生物中也具有功能[帕迪，A.B.（1989年）《科学》246，603 - 608]。为了阐明结构在该复合物功能中的作用，我们解析了T4TS的结构，作为通过诱变绘制该复合物图谱的基础。通过分子置换确定了未结合配体的酶的3.1埃结构，并对所有数据进行精修，使其达到19.9%。编码区的三个插入片段和一个缺失片段是T4TS特有的，所有位点都位于酶表面的一侧，可能编码感染周期中独特的T4特异性分子间相互作用。晶体结构通常处于未结合配体的大肠杆菌TS中所见的开放、未结合配体的构象，与封闭的三元复合物构象相反，只是至关重要的C末端插入到与Asn85残基形成氢键的活性位点，这与功能性三元复合物结构中所见的情况相同。大肠杆菌TS和T4TS之间的其他差异似乎解释了叶酰聚谷氨酸与后者结合增强的原因。
