吡咯離胺醯-tRNA合成酶酵素催化機制探討

Exploring the catalytic mechanism of Methanogenic archaeon ISO4-G1 pyrrolysyl-tRNA synthetase

鄒若主(Jo-Chu Tsou)

指導教授 ： 王彥士

國立臺灣大學/生命科學院/生化科學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202200019

摘要

生物正交胺醯-核醣核酸合成酶‧轉核醣核酸(aaRS‧tRNA)配對為擴展遺傳密碼研究中的重要元素。吡咯-轉核醣核酸合成酶(PylRS)可催化非典型胺基酸的活化，將其透過醯化作用，接至同源tRNA上。在前行研究中，演化樹內另一群不具N端結構的ΔNPylRS，已被證實在大腸桿菌與哺乳類細胞中具有生物正交性。與Methanosarcina mazei(Mm)PylRS不同的是，MmPylRS必須同時具有與tRNA互相辨識的N端，和進行催化反應的C端，而不具N端結構的ΔNPylRS，仍然可成功將非典型胺基酸嵌入蛋白中。同實驗室之前的研究顯示MmPylRS•MmtRNA配對，和Methanogenic archaeon ISO4-G1 (G1) PylRS•G1tRNA配對，為相互生物正交。在此項研究中，更進一步發現野生株Methanogenic archaeon ISO4-G1 (G1) PylRS•G1tRNA配對，G1PylRS•Methanonatronarchaeum termitum (Mt) tRNA異種配對，可拓展其酵素的受質辨認領域，包含D-胺基酸和苯丙氨酸(Phenylalanine)類似物。此外，突變株G1PylRS-GQG (其變異位被發現可擴大PylRS酵素活性位)，能辨認色氨酸(tryptophan)類似物。為了分析G1PylRS催化口袋，我們透過蛋白質晶體學，解出G1PylRS的apo form，以及含AMP、AMPCPP的複合體(bound form)晶體結構，並進一步解出G1PylRS-GQG的晶體結構。此外，確立其催化口袋介於開放和閉合間的不同過渡型態之結構，進一步暗示MmPylRS與G1PylRS之間活性位點及催化機制的不同。

關鍵字

體內嵌入非典型胺基酸 ； 吡咯-轉核醣核酸合成酶 ； 蛋白質構型改變 ； 蛋白質結晶體學

並列摘要

The bioorthogonal translation pair is a major element in the expanding genetic code research. Pyrrolysyl-tRNA synthetase (PylRS) catalyzes noncanonical amino acid (ncAA) activation and its subsequent acylation onto a cognate tRNA. ΔNPylRS, a subclass of PylRS without the N-terminal domain, was identified; its bioorthogonality was confirmed in E. coli and mammalian. Unlike the Methanosarcina mazei (Mm) PylRS, which contains a N-terminal domain (NTD) indispensable for tRNA recognition and a C-terminal domain for catalytic reaction, ΔNPylRS that lacks the N-terminal domain still can charge ncAAs efficiently. In previous study, the mutual orthogonality of MmPylRS•MmtRNA and Methanogenic archaeon ISO4-G1 (G1) PylRS•G1tRNA was established. In this study, the new substrate range of G1PylRS•G1tRNA and hybrid pair G1PylRS•Methanonatronarchaeum termitum (Mt)tRNA were found, which tunes the substrate range extending to D-noncanonical amino acids and phenylalanine analogues. Besides, the mutant, G1PylRS-GQG, in which the mutation sites were found able to enlarge the active site, showed the substrate specificity for tryptophane derivatives. The crystal structures of G1PylRS apo form, AMP bound form and AMPCPP (ATP analog) bound form were determined with counterparts for analyzing their G1PylRS catalytic pockets. The crystal structures of G1PylRS-GQG was also determined. The crystal forms adopt distinct transition states between open and closed forms revealing divergent active site arrangement and catalytic mechanism of MmPylRS and G1PylRS.

並列關鍵字

expanding genetic code ； pyrrolysyl-tRNA synthetase ； conformational changes ； protein crystallography

參考文獻

Ramakrishnan, V., Ribosome structure and the mechanism of translation. Cell 2002, 108 (4), 557-572.

Englert, M.; Moses, S.; Hohn, M.; Ling, J.; O’Donoghue, P.; Söll, D., Aminoacylation of tRNA 2′-or 3′-hydroxyl by phosphoseryl-and pyrrolysyl-tRNA synthetases. FEBS letters 2013, 587 (20), 3360-3364.

Guo, L.-T.; Wang, Y.-S.; Nakamura, A.; Eiler, D.; Kavran, J. M.; Wong, M.; Kiessling, L. L.; Steitz, T. A.; O’Donoghue, P.; Söll, D., Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proceedings of the National Academy of Sciences 2014, 111 (47), 16724-16729.

Chen, P. R.; Groff, D.; Guo, J.; Ou, W.; Cellitti, S.; Geierstanger, B. H.; Schultz, P. G., A facile system for encoding unnatural amino acids in mammalian cells. Angewandte Chemie 2009, 121 (22), 4112-4115.

Hancock, S. M.; Uprety, R.; Deiters, A.; Chin, J. W., Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. Journal of the American Chemical Society 2010, 132 (42), 14819-14824.