CRYSTAL STRUCTURE DETERMINATION OF QUEOSINE-SYNTHESIS AND OTHER TRNA MODIFICATIO

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。 我们使用SSRL束流时间对两种tRNA修饰的酶进行了结晶学研究：奎宁(Q)和苏氨酰氨基甲酰腺苷(T6A)。这是tRNA反密码子环的两个修改，改变了密码子的定义，并在翻译过程中微调核糖体功能。Q生物合成是抗志贺氏菌病抗生素的已确立的药物靶点。同样，由于其对HIV复制的要求，宿主细胞tRNA的t6A修饰已被提议作为抗HIV药物开发的潜在靶点。结合比较基因组学、生物化学和结构学方法，我们鉴定了参与Q和t6A生物合成的酶。利用SSRL X射线，我们测定了我们新发现的、也是我们在原核生物中第一个参与Q生物合成途径的酶GCYH-IB的结构，并收集了催化Q生物合成第三步的酶YkvM的天然和重原子衍生物数据。我们还测定了YrdC的晶体结构，YrdC是一种普遍保守的参与t6A生物合成的必不可少的酶，与其ATP底物形成复合体。在这次更新中，我们计划开始GCYH-IBI抑制剂复合体的结晶学研究，以及催化Q生物合成后期步骤的QueA与其tRNA底物的复合体。我们目前已经有GCYH-IB-配体络合物的晶体，后者的络合物的结晶正在进行中。我们计划继续使用SSRL的光束线自动化技术和远程访问来进行这些研究。

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. We have used SSRL beam time on this proposal to conduct crystallographic studies on enzymes responsible for the biosynthesis of two tRNA modifications: queosine (Q) and threonyl carbamoyladenosine (t6A). These are two modifications of the tRNA anticodon loop that alter codon definitions and fine tune ribosome function during translation. Q biosynthesis is an established drug target for anti-shigellosis antibiotics. Similarly, due to its requirement for HIV replication, t6A modification of host-cell tRNA has been proposed as a potential target for the development of anti-HIV pharmaceuticals. Using a combination of comparative genomic, biochemical, and structural methods, we identified enzymes involved in the biosynthesis of Q and t6A. Using SSRL x-rays, we determined the structure of GCYH-IB, our newly discovered and first enzyme in the Q biosynthesis pathway in prokaryotes, in complex with a substrate analog and we collected native and heavy-atom derivative data for YkvM, the enzyme that catalyzes the third step in Q biosynthesis. We also determined the crystal structure of YrdC, a universally conserved and essential enzyme involved in t6A biosynthesis, in complex with its ATP substrate. In this renewal, we plan to start crystallographic studies of GCYH-IBinhibitor complexes and of QueA, which catalyzes a late step in Q biosynthesis, in complex with its tRNA substrate. We currently have crystals of GCYH-IB-ligand complexes and crystallization of the latter complex is underway. We plan to continue to use the SSRL beamline automation technology and remote access for these studies.