Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification
Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification
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DOI：
10.1016/j.chembiol.2008.10.004
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发表时间：
2008-11-24
影响因子：
--
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
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作者：
Yanagisawa, Tatsuo;Ishii, Ryohei;Yokoyama, Shigeyuki
吡咯烷基-tRNA合成酶(PyIRS)将吡咯赖氨酸酯化为tRNA(Pyl)。在本研究中，N-epsilon-(叔丁氧甲酰基)-L-赖氨酸(BocLys)和N-epsilon-烯丙氧基羰基-L-赖氨酸(AlocLys)在吡咯烷酮作用下被酯化为tRNA(Pyl)。PYIRS催化片段与BocLys、ATP类似物和AlocLys-AMP络合的晶体结构表明，PYIRS需要一个带有一定大小取代基的NE-羰基。随机筛选得到的一株pYIRS(Y384F)突变体对BocLys、AlocLys和吡咯赖氨酸的体外氨基酰化和体内琥珀抑制活性均高于野生型。此外，基于Y306A结构突变的PYIRS显著提高了N-β-苄氧甲酰基-L-赖氨酸(ZLys)的体外氨酰化活性。同时具有Y306A和Y384F突变的PYIR使大规模制备含有N-epsilon-(o-叠氮苯氧羰基)-L赖氨酸(AzZLys)的蛋白质(每升培养基10 mg)成为可能。通过Staudinger连接，用荧光探针标记含有AzZLys的蛋白质。
Pyrrolysyl-tRNA synthetase (PyIRS) esterifies pyrrolysine to tRNA(PYl). In this study, N-epsilon-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N-epsilon-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA(Pyl) by PyIRS. Crystal structures of a PyIRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PyIRS requires an NE-carbonyl group bearing a substituent with a certain size. A PyIRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PyIRS. Furthermore, the structure-based Y306A mutation of PyIRS drastically increased the in vitro aminoacylation activity for N-epsilon-benzyloxycarbonyl-L-lysine (ZLys). A PyIRS with both the Y306A and Y384F mutations enabled the large-scale preparation (> 10 mg per liter medium) of proteins site-specifically containing N-epsilon-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.