6ly3
PylRS C-terminus domain mutant bound with 3-Benzothienyl-L-alanine and AMPNPPylRS C-terminus domain mutant bound with 3-Benzothienyl-L-alanine and AMPNP
Structural highlights
FunctionPYLS_METMA Catalyzes the attachment of pyrrolysine to tRNA(Pyl). Pyrrolysine is a lysine derivative encoded by the termination codon UAG (By similarity). Publication Abstract from PubMedMethanosarcina 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 A 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.tRNACUA(Pyl) pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity. Probing the Active Site of Deubiquitinase USP30 with Noncanonical Tryptophan Analogues.,Jiang HK, Wang YH, Weng JH, Kurkute P, Li CL, Lee MN, Chen PJ, Tseng HW, Tsai MD, Wang YS Biochemistry. 2020 Jun 23;59(24):2205-2209. doi: 10.1021/acs.biochem.0c00307., Epub 2020 Jun 8. PMID:32484330[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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