Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues

Author:

Taylor Christopher J.1,Hardy Florence J.1,Burke Ashleigh J.1,Bednar Riley M.2,Mehl Ryan A.2,Green Anthony P.1,Lovelock Sarah L.1ORCID

Affiliation:

1. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester Manchester UK

2. Department of Biochemistry and Biophysics Oregon State University Corvallis Oregon USA

Abstract

AbstractThe availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl‐tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3‐methyl‐L‐histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x‐ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3‐(3‐pyridyl)‐L‐alanine (3‐Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3‐Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.

Funder

UK Research and Innovation

University of Manchester

European Research Council

Biotechnology and Biological Sciences Research Council

National Science Foundation

Diamond Light Source

Publisher

Wiley

Subject

Molecular Biology,Biochemistry

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