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Mechanistic insights into tRNA thiolation catalyzed by iron-sulfur enzymes
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| Title: | Mechanistic insights into tRNA thiolation catalyzed by iron-sulfur enzymes |
| Other Titles: | 鉄硫黄クラスター含有酵素が触媒するtRNA硫黄修飾の反応機構解析 |
| Authors: | 石坂, 優人 Browse this author |
| Issue Date: | 23-Mar-2023 |
| Publisher: | Hokkaido University |
| Abstract: | tRNA transfers amino acids to ribosomes to translate genetic information into proteins. However, immature tRNA does not function immediately after transcription. Hence, tRNA undergoes post-transcriptional processes such as base/ribose modifications for maturation. To date, more than 110 base/ribose modifications have been discovered in tRNA. Particularly, thiolation (sulfur modification) is a universal and essential enzymatic reaction that improves the thermal stability and translational accuracy of tRNA.
My target is thiolation at position 54 of tRNA (5-methyl-2-thiouridine, m5s2U54), which is essential for thermophiles to survive above 70℃. m5s2U54 modification is catalyzed by 2-thiouridine synthetase TtuA with sulfur donor protein TtuB in Thermus thermophilus. Our recent structural analysis of the TtuA-TtuB complex showed that an oxygen-sensitive [4Fe-4S] cluster is required for the enzymatic activity of TtuA. Interestingly, a non-cysteine coordinated Fe (the unique Fe) of the [4Fe-4S] bound to the C-terminus of TtuB. This structure suggested that the unique Fe in TtuA relates to the sulfur transition from TtuB to tRNA, which is a novel reaction mechanism of m5s2U54 biosynthesis involving the unique Fe. On the other hand, TtuA homolog enzyme Ncs6 catalyzes thiolation at position 34 of tRNA (mcm5s2U34) with sulfur donor Urm1, which is similar to TtuA. However, a spectroscopic study indicated that Ncs6 contains [3Fe-4S], whereas crystallography supported [4Fe-4S]. Therefore, it was unclear whether the active form of tRNA-thiolation enzymes only require [4Fe-4S] or both [4Fe-4S] and [3Fe-4S].
In this study, I analyzed the structural change of Fe-S clusters in TtuA in time-course and evaluate their enzymatic activity. As a result, [3Fe-4S] spontaneously transformed into [4Fe-4S] even without the additional iron source, and the activity of TtuA gradually recovered corresponding to an increase in [4Fe-4S]. I also revealed that [3Fe-4S]-TtuA cannot bind to the C-terminus of TtuB, indicating that only [4Fe-4S]-TtuA is an active form. Furthermore, I found that TtuB does not release sulfur until tRNA is activated by ATP, and identified the critical residues of TtuA. Considering the similarity, I proposed that [4Fe-4S] is generally an active form in tRNA-thiolation enzymes and the detailed tRNA-thiolation mechanism catalyzed by TtuA. These findings showed that the time-course analysis of the structure and activity under strictly anaerobic conditions is necessary to elucidate the reaction mechanism of enzymes containing Fe-S clusters. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第15305号 |
| Degree Level: | 博士 |
| Degree Discipline: | 生命科学 |
| Examination Committee Members: | (主査) 教授 尾瀬 農之, 教授 相沢 智康, 教授 比能 洋 |
| Degree Affiliation: | 生命科学院（生命科学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/89647 |
| Appears in Collections: | 課程博士 (Doctorate by way of Advanced Course) > 生命科学院(Graduate School of Life Science)
学位論文 (Theses) > 博士 （生命科学）
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