STRUCTURAL STUDIES OF TSCP AND ITS MUTANTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 海栖热袍菌的thy 1基因编码一种胸苷酸合成酶互补蛋白（TSCP），即TM 0449。 TSCP在缺乏胸苷酸外部来源的情况下与细胞存活有关。一般来说，TSCP？补充胸苷酸合成酶（TS）的活性。 胸苷酸合成是dTMP的唯一从头合成途径中的终末步骤。 因此，TS抑制停止DNA产生，阻止细胞周期并最终导致细胞死亡。TSCP家族与经典TS没有结构同源性序列。 虽然在真核生物中非常罕见，但TSCP基因广泛分布于细菌的生命领域。 TSCP家族的许多成员是人类致病菌。 在继续我们的TM 0449的结构研究，我们已经研究了12个突变体的结构，旨在了解酶的功能方面。 对三个双突变体（F158 G-W160 A、F158 A-W160 A、F158 A-F160 Q）与底物和辅因子的结构研究有助于理解酶催化的机理方面。 该项目目前的重点是利用炭疽、斑疹伤寒和结核病病原体的酶来探索TSCP的机制。我们刚刚开始从立克次体中结晶TSCP酶。 不同生物体中酶的结构有助于我们了解重要的功能基团，并为设计抗肿瘤药物提供可能性。 我们在厌氧条件下捕获中间体的结构研究也在进行中。 在厌氧条件下制备的两种复合物的初始结构表明，FAD的黄素环在FAD-酶复合物以及FAD-酶-dUMP复合物中是无序的。 因此，我们现在采用更好的化学方法来捕获活性部位的叶酸。 与Amnon Kohen教授的新合作？他的研究小组在复杂合成、厌氧实验和结晶方面提供了许多机理性的见解。

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.