Volume 252, 2024

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

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.

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

  • This article is part of the themed collection: Biocatalysis

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Article information

Article type
Paper
Submitted
02 feb 2024
Accepted
16 feb 2024
First published
07 mar 2024
This article is Open Access
Creative Commons BY license

Faraday Discuss., 2024,252, 295-305

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

A. E. Hutton, J. Foster, J. E. J. Sanders, C. J. Taylor, S. A. Hoffmann, Y. Cai, S. L. Lovelock and A. P. Green, Faraday Discuss., 2024, 252, 295 DOI: 10.1039/D4FD00019F

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