Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNA^Sec , which is subsequently converted by selenocysteine synthase to Sec-tRNA^Sec . Escherichia coli SerRS serylates tRNA^Sec poorly; this may hinder efficient production of designer selenoproteins in vivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNA^Sec synthesis. They display 10-, 8-, and 4-fold increased k_cat /K_M values compared to wild-type SerRS using synthetic tRNA^Sec species as substrates. The enzyme variants also facilitate in vivo read-through of a UAG codon in the position of the critical serine₁₄₆ of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.