An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes

Amy E. Hutton, Jake Foster, James E. J. Sanders, Christopher J. Taylor, Stefan A. Hoffmann, Yizhi Cai, Sarah L. Lovelock, Anthony Green

Research output: Contribution to journalArticlepeer-review

Abstract

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Original languageEnglish
Pages (from-to)295-305
Number of pages11
JournalFaraday Discussions
Volume252
Early online date7 Mar 2024
DOIs
Publication statusPublished - 11 Sept 2024

Keywords

  • Amino Acyl-tRNA Synthetases/metabolism
  • Methylhistidines/metabolism
  • Lysine/chemistry
  • Catalytic Domain
  • Histidine/chemistry

Research Beacons, Institutes and Platforms

  • Dalton Nuclear Institute

Fingerprint

Dive into the research topics of 'An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes'. Together they form a unique fingerprint.

Cite this