The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme

[Display omitted] •Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) activity was enhanced when coupled with Ca. Methanomethylphilus alvus tRNAPyl (CmaPylT).•CmaPylT enhance MmPylRS activity by stabilizing full length MmPylRS.•CmaPylT prevents MmPylRS from cleavage after A189.•Introducing P188 mutations to MmPylRS generally improves activity of MmPylRS variants.•This discovery will be generally useful to develop systems for enhanced incorporation of noncanonical amino acids at amber codon. The amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique has been widely used in both basic and applied research. So far more than two hundred ncAAs have been genetically encoded by amber codon in both prokaryotes and eukaryotes using wild-type and engineered pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl (PylT) pairs. Methanosarcina mazei PylRS (MmPylRS) is arguably one of two most used PylRS variants. However, it contains an unstable N-terminal domain that is usually cleaved from the full-length protein during expression and therefore leads to a low enzyme activity. We discovered that the cleavage takes place after A189 and this cleavage is inhibited when MmPylRS is co-expressed with Ca. Methanomethylophilus alvus tRNAPyl (CmaPylT). In the presence of CmaPylT, MmPylRS is cleaved after an alternative site K110. MmPylRS is active toward CmaPylT. Its combined use with CmaPylT leads to enhanced incorporation of Nε-Boc-lysine (BocK) at amber codon. To prevent MmPylRS from cleavage after A189 in the presence of its cognate M. mazei tRNAPyl (MmPylT), we introduced mutations at P188. Our results indicated that the P188G mutation stabilizes MmPylRS. We showed that the P188G mutation in wild-type MmPylRS or its engineered variants allows enhanced incorporation of BocK and other noncanonical amino acids including Nε-acetyl-lysine when they are co-expressed with MmPylT.