Abstract
Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus 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 lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs 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 Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.
Highlights
Pyrrolysyl-tRNA synthetase has been applied for expanding the repertoire of genetically encoded amino acids [1,2,3,4]
Each of CMaPylRS, MmPylRS, MbPylRS, and DhPylSc was expressed from a lactose-inducible promoter in the E. coli BL21-Gold(DE3) strain together with one of tRNAPyl molecules from D. hafniense, M. mazei, and Ca
Since the base sequence of tRNAPyl is identical between certain strains of M. mazei and M. barkeri and M. mazei tRNAPyl designated as Mm tRNAPyl was used as a common substrate for MmPylRS and MbPylRS
Summary
Pyrrolysyl-tRNA synthetase has been applied for expanding the repertoire of genetically encoded amino acids [1,2,3,4]. PylRS naturally occurs in archaea and certain bacteria species attaching pyrrolysine to tRNAPyl to translate the UAG amber stop codon [1,5]. The utility of PylRS is based on the following observations. This pair does not cross-react with the aminoacyl-tRNA synthetase (aaRS)—tRNA pairs of host cells such as Escherichia coli and mammalian cells. The noncanonical secondary structure of tRNAPyl , which is a small D loop and an extended anticodon stem, apparently underlines this orthogonality [6]. Since pyrrolysine is not included in the metabolic pathways of host cells, the promiscuous incorporation of pyrrolysine and its derivatives into proteins is avoided even when
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