STRUCTURAL STUDIES OF TSCP AND ITS MUTANTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该基因编码胸苷合成酶互补蛋白(TSCP)TM0449。在缺乏外源胸苷的情况下，TSCP已被认为与细胞存活有关。总体而言，通心络、S对胸苷合成酶(TS)的活性起补充作用。胸苷合成是合成DTMP的唯一从头合成途径的最后一步。因此，抑制TS会阻止DNA的产生，阻止细胞周期，最终导致细胞死亡。TSCP家族与经典TS没有结构同源性的序列。虽然TSCP基因在真核生物中极为罕见，但在生命的细菌领域中分布广泛。TSCP家族的许多成员都是人类致病菌。在我们对TM0449的结构研究的基础上，我们研究了12个突变体的结构，旨在了解该酶的功能方面。对三个双突变体(F158G-W160A、F158A-W160A、F158A-F160Q)及其底物和辅因子的结构研究有助于了解酶催化的机理。该项目目前的重点是利用引起炭疽、斑疹伤寒和结核病的病原体的酶来探索TSCP的机制。我们刚刚开始了立克次体TSCP酶的结晶。来自不同生物体的酶的结构将帮助我们了解重要的功能基团和设计抗生素药物的可能性。我们在厌氧条件下捕获中间体的结构研究也在进行中。其中两个在厌氧条件下制备的配合物的初步结构表明，FAD的黄素环在FAD-酶复合体和FAD-酶-转储复合体中是无序的。因此，我们现在正在使用更好的化学方法来捕获活性部位中的叶酸。与安农？科恩？S教授的新合作为复杂的合成、厌氧实验和结晶提供了许多机械方面的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The thy1 gene of Thermotoga maritima encodes a thymidylate synthase complementing protein (TSCP), TM0449. TSCPs have been implicated in cell survival in the absence of external sources of thymidylate. In general, TSCP?s complement the activity of thymidylate synthase (TS). Thymidylate synthesis is the terminal step in the sole de novo synthetic pathway to dTMP. Consequently, TS inhibition stops DNA production, arresting the cell cycle and eventually leading to cell death. The TSCP family share no sequence of structural homology to classical TS. Although extremely rare in Eukaryotes the TSCP gene is widely distributed within the bacterial domain of life. Many members of the TSCP family are human pathogenic bacteria. In continuation with our structural study of TM0449, we have studied the structures of 12 muitants that are aimed at understanding the functional aspects of the enzyme. The structural study of the three double mutants (F158G-W160A,F158A-W160A, F158A-F160Q) with substrates and cofactors helps to understand the mechanistic aspects of the enzyme catalysis. The current focus of the project is to explore the mechanism of TSCP using enzymes from pathogens causing Anthrax, Typhus, and Tuberculosis. We have just started the crystallization of the TSCP enzyme from Rickettsia. The structures of enzymes from different organisms will help us understand the important functional groups and the possibility of the design of an antibiotic-drug. Our structural study to trap the intermediates under anaerobic conditions is also in progress. The initial structures from two of these complexes prepapred under anerobic conditions show that the flavine ring of the FAD is disordered in the FAD-enzyme complex as well as the FAD-enzyme-dUMP complex. Therefore, we are now employing better chemical procedures to trap the folate in the active site. The new collaboration with Professor Amnon Kohen?s group provides lot of mechanistic insights to the complex synthesis, anaerobic experiments, and crystallization.