https://scholar.gist.ac.kr/handle/local/7941 2026-05-13T15:11:40Z https://scholar.gist.ac.kr/handle/local/32982 Title: Wobble Uridine C5 Modification in Escherichia coli tRNA Author(s): Jaehun Yoo Abstract: The post-transcriptional modification of the wobble uridine in E. coli tRNAs is significant for facilitating non-Watson-Crick base pairing which modulates decoding capabilities. One of the common wobble uridine modification, uridine 5-oxyacetic acid (or also called as 5-carboxymethoxyuridine, cmo5U) biogenesis via 5-hydroxyuridine (ho5U) formation is widely conserved in gram negative bacteria. tRNA bound enzyme structure determination by X-ray crystal structures of wobble uridine hydroxylases YceA (O2 dependent), YegQ (prephenate dependent), and carboxymethyltransferase CmoB from E. coli have been attempted to elucidate the unknown chemical mechanism. The in vitro activity of YceA to conduct reaction on stem loop tRNAAla(UGC), tRNAPro(UGG), and tRNAThr(UGU) was confirmed by LC-MS analysis. Similarly, CmoB coupled assay was carried out with YceA which generated cmo5U in full length and stem loop tRNA. Overall, the YceA and CmoB activity has been reconstituted. The requirements for both enzymes’ substrate specificity was noted while details remain elusive, including the reaction mechanism of YceA. 2019-12-31T15:00:00Z https://scholar.gist.ac.kr/handle/local/33284 Title: Versatile small molecule kinase assay through real-time, ratiometric fluorescent monitoring of interchange for ATP and ADP based on pyrene-Zn2+-DPA complex Author(s): Jihoon Kim Abstract: Since kinase, which induces the transfer of phosphate group, is associated with a biological process and various diseases, kinase has been recently identified as a major target in drug discovery and its activity measurement is of crucial importance. Herein, we introduced a versatile and real-time kinase assay platform using pyrene-based ratiometric fluorescence probe to monitor small molecule kinase activity. The fluorescent probe, pyrene-Zn2+-DPA complex, has a characteristic showing distinct excimer and monomer fluorescence according to the changed ratio of ATP and ADP for the kinase progression. This system was adapted to the various enzymes as shown by utilizing for the hexokinase and the creatine kinase which phosphorylates and dephosphorylates each substrate, respectively, and the quantitative analysis of each well-known inhibitor was estimated. Moreover, it could be monitored by the naked eye with a simple light source, indicating a powerful tool for inhibitor assay. As a result, the proposed assay system can be extended as a general platform applicable to various kinases and suitable for high throughput screening methods for inhibitor screening. 2020-12-31T15:00:00Z https://scholar.gist.ac.kr/handle/local/33415 Title: Various structures of CmoB provide structural insight into cmo5U modification mechanism Author(s): Sehwang Jeong Abstract: The 34th uridine of tRNA is always almost modified in three domains of life. The 5-carboxymethoxyuridiene (cmo5U), which is one of the species of uridine modification, is generated by carboxymethyl transfer enzyme CmoB in gram-negative bacteria. Unique metabolite Carboxy-S-adenosyl-l-methionine (CxSAM) is used for carboxymethyl donor of CmoB reaction and S-adenosyl-l-homocysteine (SAH) is a co-product of the transfer reaction. In this study, I present four X-ray crystal structures of CmoB from Vibrio vulnificus; i.e., apo-, SAH bound, CxSAM bound and tRNA bound forms. The crystal structure of apo-CmoB displays novel open conformation of the protein, which has not been observed in previously reported structures. Flexible loop region is stabilized with neighboring symmetry related molecules. The Crystal structures of CmoB bound with SAH and CxSAM provide molecular basis for cofactor selectivity. In addition, binding assays for SAH with CmoB was conducted to investigate the affinity for the cofactor in solution. Furthermore, a co-crystal structure of tRNA-CmoB complex has been determined for the first time in this study. However, the structure appears to represent an inactive form, where the wobble uridine is in stacked pose and too distant from the bound CxSAM for the transfer reaction to occur. The mutagenesis assay suggests that the amino acid residues Arg-90, Arg-242, Arg-247 and Asn-248 are important to recognizing phosphate backbone of tRNA, validating that the structure provides a valuable information on the molecular interactions between tRNA and the enzyme in forming the pre-Michaelis complex. 2020-12-31T15:00:00Z https://scholar.gist.ac.kr/handle/local/33282 Title: Utilization of Pyridylamido-based Ligand System for Synthesis of Hafnium and Tungsten Complexes Author(s): Donghyeon Kim Abstract: Complexes having early transition metal like Ti, Zr, and Hf are widely used in olefin polymerization as catalysts. These complexes have been actively researched since the invention of Ziegler-natta catalysts. And in 2007, Dow and symyx co-researched asymmetric structure based hafnium catalysts which have high activity and high isoselectivity in α-olefin polymerization. The structural features of this hafnium catalyst are pyridine nitrogen, amido nitrogen, aryl bond carbon and two methyl groups coordinating hafnium metal center. Because of this sterically surrounded system and asymmetric structure, hafnium complex has restricted size of active site and insertion direction of monomers. Although this pyridyl-amido complex has good catalytic activity in olefin polymerization, it has short catalytic lifetimes (< 50 minutes). For overcoming these short catalytic lifetimes, we tried to modifying ligand system. In our research, we synthesized ligand scaffold through organic synthesis and then proceeded metalation for hafnium complex. After synthesizing, we analyze the structure by NMR spectroscopy. And we confirmed whether the synthesized complex has catalytic activity in olefin polymerization through the copolymerization test of the synthesized complex. And then, we synthesized a metal complex by reacting with another metal (W) using the synthesized ligand and tried to confirm the reactivity through electrochemistry of the synthesized tungsten complex. 2020-12-31T15:00:00Z