STRUCTURAL STUDIES OF TM

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。子项目和 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。 在继续我们对TM 0449的结构研究中，我们现在已经收集了大约12种酶突变体的衍射数据。 这些突变体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。 目前的研究集中在制备复合物和在厌氧气氛中结晶它们。 我们已经在厌氧条件下生长了两个突变体的晶体，结构研究正在进行中。在少量氧存在下还原的FAD快速转化为氧化形式是使具有还原形式的FAD的复合物结晶的主要问题。 活性位点中不存在FAD结合还原形式的结构。在厌氧条件下制备的配合物的结构信息将是有价值的TSCP催化机理的研究。

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 now collected diffraction data for around 12 mutants of the enzyme. These mutants 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 study is focused on preparing the complexes and crystallizing them in an anaerobic atmosphere. We have grown crystals of two mutants under anaerobic conditions and the structural study is in progress. The rapid conversion of the reduced FAD to the oxidized form in the presence of small amounts of oxygen is a major problem in crystallizing complexes with the reduced form of FAD. There are no structures available with the reduced form of FAD bound in the active site. The structural information derived from the complexes prepared under anaerobic conditions will be valuable for the mechanistic study of the TSCP catalysis.