Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes
2018
Genetic codeexpansion has largely relied on two types of the tRNA—
aminoacyl-tRNA synthetasepairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various
lysinederivatives into proteins. The widely used PylRS from
Methanosarcinaceaecomprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from
CandidatusMethanomethylophilus alvus (CMaPylRS) is a
single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate
lysinederivatives much more efficiently (up to 14-times) than
MethanosarcinaceaePylRSs in Escherichia coli cell-based and
cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural
idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Ne-benzyloxycarbonyl-l-
lysine(ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of
lysinederivatives in
expanded codes.
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