Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

Hsueh-Wei Tseng,Nediljko Budisa,Huan Sun,Tobias Baumann,Zoya Ignatova,Yane-Shih Wang

doi:10.3390/molecules25194418

Abstract

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

Highlights

IntroductionNon-canonical amino acids (ncAAs) can be incorporated into recombinant proteins using a number of in vivo and in vitro methods [1,2]
Protein translation is a process by which a protein sequence is being built up according to the information provided in the messenger RNA
Based on the Pyrrolysyl–tRNA synthetase (PylRS) structure and activity data collected from a large number of enzyme mutants, residues V401 and W417 (Figure 2) were targeted

Summary

Introduction

Non-canonical amino acids (ncAAs) can be incorporated into recombinant proteins using a number of in vivo and in vitro methods [1,2]. These non-natural insertions provide useful modifications that might take place on the protein backbone or on the amino acids’ side chains. Other protein backbone modifications are typically produced using in vitro ribosomal translation systems. Β-, or γ-amino acids can be inserted into target polypeptide sequences by using engineered in vitro translation systems [1]. Α-hydroxy acids can be incorporated into proteins, for example in response to in-frame amber codons via the action of specific engineered systems [7]. A typical example in nature is the rare 22nd proteinogenic amino acid pyrrolysine (Pyl), which appears in very few proteomes of methanogenic microorganisms [2]

Methods

Results

Discussion

Conclusion