Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7–2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS·tRNA_CUA^Pyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.

中文翻译：

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用非规范色氨酸类似物探查去泛素酶USP30的活性位点。

马氏甲烷八叠球菌吡咯基-tRNA合成酶（PylRS）及其相关的tRNA已进化为产生遗传编码的非规范氨基酸（ncAAs）。带有吡咯环修饰的色氨酸（Trp）类似物在酶活性和底物特异性方面的空间和极性调节方面的应用仍然受到限制。在本文中，我们报道了进化的PylRS FOWRS2在将五个Trp类似物有效掺入去泛素化酶USP30中的应用中，以破译W475对泛素选择性的作用。五个FOWRS-C / Trp类似物复合物的结构在1.7–2.5Å分辨率下显示出多种ncAA结合模式。USP30活性位点附近的W475被Trp类似物取代，并检查了对活性的影响以及对泛素连接类型的选择性。1-丙氨酸诱导了独特的K6-特异性。集体地，进化的PylRS· tRNA _CUA ^Pyl对通过遗传编码的非经典Trp类似物揭示了USP30-W475在其泛素选择性中的空间作用。