藉由分析三個域中的Prolyl-tRNA synthetase (ProRS)可以歸納出兩種 不同的結構，真核生物/古生菌類型(E-type) ProRS和原核生物類型(P-type) ProRS，前者在C-terminal多一段domain，後者在motif 2和motif 3之間有一 段額外的domain。除此之外，不同ProRS辨識的tRNAPro也有所不同，ProRS主要 會辨識tRNAPro的anticodon和acceptor stem，雖然anticodon在所有生物中都非 常保守，acceptor stem上的序列卻不太一樣，在細菌中是G72/A73，但在真核 生物中是C72/C73，有趣的是，E. coli tRNAPro中的G72/A73是主要被辨識的序 列，但在人類tRNAPro中C72/C73並不被辨識。令人訝異的是，有數種細菌被發現 帶有E-type而非P-type的ProRS，例如嗜熱菌Thermus thermophilus，我們的 實驗結果顯示這種細菌的E-type ProRS能夠辨識G72/A73，這顯示了T. thermophilus ProRS (TtProRS)儘管在結構上類似人類的ProRS，辨識的特性 卻類似E. coli ProRS，會同時辨認anticodon上的G35/G36，和acceptor stem 上的G72/A73。然而，不同於典型P-type ProRS，TtProRS並不使用在motif 2 上非常保守的R胺基酸，而是使用同樣在motif 2中的RTR 序列，然而在典型的 E-type ProRS中RTR並不保守，這說明TtProRS從典型E-type發展出獨特的序列 及辨識機制。此外，我們也發現TtProRS相對於其他E-type ProRS對 halofuginone (一種從febrifugine衍生而來的E-type ProRS抑制劑，結構類似 於Pro-A76)耐受性較高，特性比較接近細菌類型的ProRS。這項研究顯示了一 個不可或缺且非常保守的酵素能隨著環境變化適應新的受質。

Analysis of prolyl-tRNA synthetase (ProRS) from three domains of life uncovers two separate architectures: a eukaryote/archaeon-like (E-type) ProRS, distinguished by a C-terminal extension domain, and a prokaryote-like (P-type) ProRS, distinguished by an insertion domain. Diversity also appears in tRNAPro as the substrate of ProRS. While the anticodon elements of tRNAPro are highly conserved among all organisms and important for aminoacylation, the acceptor stem elements have diverged, with G72/A73 conserved in bacteria and C72/C73 conserved in eukaryotes. In E. coli tRNAPro, G72/A73 are major determinants, whereas in humans, C72/C73 are dispensable. Paradoxically, several bacteria have been revealed to possess an E-type ProRS, with one example being Thermus thermophilus ProRS (TtProRS). This bacterial E-type ProRS has been reported to selectively charge the P- type tRNAPro with G72/A73. This investigation reveals that despite the structural similarity with human ProRS, TtProRS maintains a strong recognition towards the anticodon elements G35/G36 and the acceptor-stem elements G72/A73, similar to E. coli ProRS. However, instead of relying on the strictly conserved R residue in the motif 2 loop of P-type ProRS to recognize G72/A73, TtProRS accomplishes this using RTR, a divergent sequence found within the E-type ProRS motif 2 loop. Here we also demonstrate TtProRS's relatively resistance to halofuginone, a synthetic inhibitor of eukaryote-type ProRS derived from febrifugine mimicking Pro-A76, a characteristic similar to that of bacterium-type ProRS. This study highlights the adaptability of a usually conserved essential enzyme to adjust to a new substrate.

An, S., Barany, G., & Musier-Forsyth, K. (2008). Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase. Nucleic Acids Res, 36(8), 2514-2521. https://doi.org/10.1093/nar/gkn063
Antika, T. R., Chrestella, D. J., Ivanesthi, I. R., Rida, G. R. N., Chen, K. Y., Liu, F. G., Lee, Y. C., Chen, Y. W., Tseng, Y. K., & Wang, C. C. (2022). Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla. Nucleic Acids Res, 50(4), 2190-2200. https://doi.org/10.1093/nar/gkac026
Antika, T. R., Chrestella, D. J., Tseng, Y. K., Yeh, Y. H., Hsiao, C. D., & Wang, C. C. (2023). A naturally occurring mini-alanyl-tRNA synthetase. Commun Biol, 6(1), 314. https://doi.org/10.1038/s42003-023-04699-0
Ardell, D. H. (2010). Computational analysis of tRNA identity. FEBS Lett, 584(2), 325-333. https://doi.org/10.1016/j.febslet.2009.11.084
Bartholow, T. G., Sanford, B. L., Cao, B., Schmit, H. L., Johnson, J. M., Meitzner, J., Bhattacharyya, S., Musier-Forsyth, K., & Hati, S. (2014). Strictly conserved lysine of prolyl-tRNA Synthetase editing domain facilitates binding and positioning of misacylated tRNA(Pro.). Biochemistry, 53(6), 1059-1068. https://doi.org/10.1021/bi401279r
Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., & Cullin, C. (1993). A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res, 21(14), 3329-3330. https://doi.org/10.1093/nar/21.14.3329
Berthonneau, E., & Mirande, M. (2000). A gene fusion event in the evolution of aminoacyl-tRNA synthetases. FEBS Lett, 470(3), 300-304. https://doi.org/10.1016/s0014-5793(00)01343-0
Beuning, P. J., & Musier-Forsyth, K. (2001). Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase. J Biol Chem, 276(33), 30779-30785. https://doi.org/10.1074/jbc.M104761200
Boeke, J. D., Trueheart, J., Natsoulis, G., & Fink, G. R. (1987). 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol, 154, 164-175. https://www.ncbi.nlm.nih.gov/pubmed/3323810
Burbaum, J. J., & Schimmel, P. (1991). Structural relationships and the classification of aminoacyl-tRNA synthetases. J Biol Chem, 266(26), 16965-16968.
Burke, B., Lipman, R. S., Shiba, K., Musier-Forsyth, K., & Hou, Y. M. (2001). Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase. J Biol Chem, 276(23), 20286-20291. https://doi.org/10.1074/jbc.m100456200
Burke, B., Yang, F., Chen, F., Stehlin, C., Chan, B., & Musier-Forsyth, K. (2000). Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system. Biochemistry, 39(50), 15540-15547. https://doi.org/10.1021/bi001835p
Carter, C. W., Jr. (1993). Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu Rev Biochem, 62, 715-748. https://doi.org/10.1146/annurev.bi.62.070193.003435
Cavarelli, J., Rees, B., Ruff, M., Thierry, J. C., & Moras, D. (1993). Yeast tRNA(Asp) recognition by its cognate class II aminoacyl-tRNA synthetase. Nature, 362(6416), 181-184. https://doi.org/10.1038/362181a0
Chang, C. P., Lin, G., Chen, S. J., Chiu, W. C., Chen, W. H., & Wang, C. C. (2008). Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain. J Biol Chem, 283(45), 30699-30706. https://doi.org/10.1074/jbc.M805339200
Chang, C. Y., Chien, C. I., Chang, C. P., Lin, B. C., & Wang, C. C. (2016). A WHEP Domain Regulates the Dynamic Structure and Activity of Caenorhabditis elegans Glycyl-tRNA Synthetase. J Biol Chem, 291(32), 16567-16575. https://doi.org/10.1074/jbc.M116.730812
Chang, K. J., Lin, G., Men, L. C., & Wang, C. C. (2006). Redundancy of non-AUG initiators. A clever mechanism to enhance the efficiency of translation in yeast. J Biol Chem, 281(12), 7775-7783. https://doi.org/10.1074/jbc.M511265200
Crepin, T., Yaremchuk, A., Tukalo, M., & Cusack, S. (2006). Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain. Structure, 14(10), 1511-1525. https://doi.org/10.1016/j.str.2006.08.007
Cusack, S. (1993). Sequence, structure and evolutionary relationships between class 2 aminoacyl-tRNA synthetases: an update. Biochimie, 75(12), 1077-1081. https://doi.org/10.1016/0300-9084(93)90006-e
Cusack, S., Yaremchuk, A., Krikliviy, I., & Tukalo, M. (1998). tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase. Structure, 6(1), 101-108. https://doi.org/10.1016/s0969-2126(98)00011-2
Eriani, G., Delarue, M., Poch, O., Gangloff, J., & Moras, D. (1990). Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature, 347(6289), 203-206. https://doi.org/10.1038/347203a0
Eswarappa, S. M., Potdar, A. A., Sahoo, S., Sankar, S., & Fox, P. L. (2018). Metabolic origin of the fused aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase. J Biol Chem, 293(49), 19148-19156. https://doi.org/10.1074/jbc.RA118.004276
Giegé, R., & Eriani, G. (2023). The tRNA identity landscape for aminoacylation and beyond. Nucleic Acids Res, 51(4), 1528-1570. https://doi.org/10.1093/nar/gkad007
Hati, S., Ziervogel, B., Sternjohn, J., Wong, F. C., Nagan, M. C., Rosen, A. E., Siliciano, P. G., Chihade, J. W., & Musier-Forsyth, K. (2006). Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids. J Biol Chem, 281(38), 27862-27872. https://doi.org/10.1074/jbc.M605856200
Himeno, H., Hasegawa, T., Ueda, T., Watanabe, K., Miura, K., & Shimizu, M. (1989). Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation. Nucleic Acids Res, 17(19), 7855-7863. https://doi.org/10.1093/nar/17.19.7855
Hou, Y. M., & Schimmel, P. (1988). A simple structural feature is a major determinant of the identity of a transfer RNA. Nature, 333(6169), 140-145. https://doi.org/10.1038/333140a0
Ibba, M., & Soll, D. (2000). Aminoacyl-tRNA synthesis. Annu Rev Biochem, 69, 617-650. https://doi.org/10.1146/annurev.biochem.69.1.617
Ivanesthi, I. R., Rida, G. R. N., Setiawibawa, A. A., Tseng, Y. K., Muammar, A., & Wang, C. C. (2023). Recognition of tRNA(His) in an RNase P-Free Nanoarchaeum. Microbiol Spectr, 11(2), e0462122. https://doi.org/10.1128/spectrum.04621-22
Jain, V., Yogavel, M., Oshima, Y., Kikuchi, H., Touquet, B., Hakimi, M. A., & Sharma, A. (2015). Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis. Structure, 23(5), 819-829. https://doi.org/10.1016/j.str.2015.02.011
Keller, T. L., Zocco, D., Sundrud, M. S., Hendrick, M., Edenius, M., Yum, J., Kim, Y. J., Lee, H. K., Cortese, J. F., Wirth, D. F., Dignam, J. D., Rao, A., Yeo, C. Y., Mazitschek, R., & Whitman, M. (2012). Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol, 8(3), 311-317. https://doi.org/10.1038/nchembio.790
Kwon, N. H., Fox, P. L., & Kim, S. (2019). Aminoacyl-tRNA synthetases as therapeutic targets. Nat Rev Drug Discov, 18(8), 629-650. https://doi.org/10.1038/s41573-019-0026-3
Lee, Y. H., Chang, C. P., Cheng, Y. J., Kuo, Y. Y., Lin, Y. S., & Wang, C. C. (2017). Evolutionary gain of highly divergent tRNA specificities by two isoforms of human histidyl-tRNA synthetase. Cell Mol Life Sci, 74(14), 2663-2677. https://doi.org/10.1007/s00018-017-2491-3
Lee, Y. H., Lo, Y. T., Chang, C. P., Yeh, C. S., Chang, T. H., Chen, Y. W., Tseng, Y. K., & Wang, C. C. (2019). Naturally occurring dual recognition of tRNA(His) substrates with and without a universal identity element. RNA Biol, 16(9), 1275-1285. https://doi.org/10.1080/15476286.2019.1626663
Liu, H., & Musier-Forsyth, K. (1994). Escherichia coli proline tRNA synthetase is sensitive to changes in the core region of tRNA(Pro). Biochemistry, 33(42), 12708-12714. https://doi.org/10.1021/bi00208a023
Liu, H., Peterson, R., Kessler, J., & Musier-Forsyth, K. (1995). Molecular recognition of tRNA(Pro) by Escherichia coli proline tRNA synthetase in vitro. Nucleic Acids Res, 23(1), 165-169. https://doi.org/10.1093/nar/23.1.165
Manickam, Y., Malhotra, N., Mishra, S., Babbar, P., Dusane, A., Laleu, B., Bellini, V., Hakimi, M. A., Bougdour, A., & Sharma, A. (2022). Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development. PLoS Pathog, 18(3), e1010363. https://doi.org/10.1371/journal.ppat.1010363
Ohtani, N., Tomita, M., & Itaya, M. (2010). An extreme thermophile, Thermus thermophilus, is a polyploid bacterium. J Bacteriol, 192(20), 5499-5505. https://doi.org/10.1128/jb.00662-10
Oshima, T., & Imahori, K. (1974). Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa. International Journal of Systematic and Evolutionary Microbiology, 24(1), 102-112. https://doi.org/https://doi.org/10.1099/00207713-24-1-102
Pena, N., Dranow, D. M., Hu, Y., Escamilla, Y., & Bullard, J. M. (2019). Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform. Protein Sci, 28(4), 727-737. https://doi.org/10.1002/pro.3579
Pino, P., Aeby, E., Foth, B. J., Sheiner, L., Soldati, T., Schneider, A., & Soldati-Favre, D. (2010). Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation in Apicomplexa. Mol Microbiol, 76(3), 706-718. https://doi.org/10.1111/j.1365-2958.2010.07128.x
Rajendran, V., Kalita, P., Shukla, H., Kumar, A., & Tripathi, T. (2018). Aminoacyl-tRNA synthetases: Structure, function, and drug discovery. Int J Biol Macromol, 111, 400-414. https://doi.org/10.1016/j.ijbiomac.2017.12.157
Ruff, M., Krishnaswamy, S., Boeglin, M., Poterszman, A., Mitschler, A., Podjarny, A., Rees, B., Thierry, J. C., & Moras, D. (1991). Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science, 252(5013), 1682-1689. https://doi.org/10.1126/science.2047877
Sankaranarayanan, R., Dock-Bregeon, A. C., Romby, P., Caillet, J., Springer, M., Rees, B., Ehresmann, C., Ehresmann, B., & Moras, D. (1999). The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site. Cell, 97(3), 371-381. https://doi.org/10.1016/s0092-8674(00)80746-1
Stehlin, C., Burke, B., Yang, F., Liu, H., Shiba, K., & Musier-Forsyth, K. (1998). Species-specific differences in the operational RNA code for aminoacylation of tRNAPro. Biochemistry, 37(23), 8605-8613. https://doi.org/10.1021/bi980364s
SternJohn, J., Hati, S., Siliciano, P. G., & Musier-Forsyth, K. (2007). Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain. Proc Natl Acad Sci U S A, 104(7), 2127-2132. https://doi.org/10.1073/pnas.0611110104
Tang, H. L., Yeh, L. S., Chen, N. K., Ripmaster, T., Schimmel, P., & Wang, C. C. (2004). Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J Biol Chem, 279(48), 49656-49663. https://doi.org/10.1074/jbc.M408081200
Tye, M. A., Payne, N. C., Johansson, C., Singh, K., Santos, S. A., Fagbami, L., Pant, A., Sylvester, K., Luth, M. R., Marques, S., Whitman, M., Mota, M. M., Winzeler, E. A., Lukens, A. K., Derbyshire, E. R., Oppermann, U., Wirth, D. F., & Mazitschek, R. (2022). Elucidating the path to Plasmodium prolyl-tRNA synthetase inhibitors that overcome halofuginone resistance. Nat Commun, 13(1), 4976. https://doi.org/10.1038/s41467-022-32630-4
Vargas-Rodriguez, O., & Musier-Forsyth, K. (2013). Exclusive use of trans-editing domains prevents proline mistranslation. J Biol Chem, 288(20), 14391-14399. https://doi.org/10.1074/jbc.M113.467795
Yaremchuk, A., Cusack, S., & Tukalo, M. (2000). Crystal structure of a eukaryote/archaeon-like protyl-tRNA synthetase and its complex with tRNAPro(CGG). Embo j, 19(17), 4745-4758. https://doi.org/10.1093/emboj/19.17.4745
Yaremchuk, A. D., Sergeevich, B., & Tukalo, M. (2012). Prolyl-tRNA synthetase from Thermus thermophilus is eukaryotic-like but aminoacylates prokaryotic tRNAPro. Biopolymers and Cell, 28, 434-440. https://doi.org/10.7124/bc.000133
Zhang, C. M., & Hou, Y. M. (2004). Synthesis of cysteinyl-tRNACys by a prolyl-tRNA synthetase. RNA Biol, 1(1), 35-41.