Engineering Translation Components Improve Incorporation of Exotic Amino Acids.

Takayuki Katoh,Hiroaki Suga

doi:10.3390/ijms20030522

Abstract

Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.

Highlights

In endogenous ribosomal translation, only the 20 proteinogenic amino acids (PAAs), the 19 L-α-amino acids, and glycine are available as building blocks for peptide/protein synthesis
The efficiencies of ribosomal incorporation of nonproteinogenic amino acids (nPAAs) vary depending on their chemical structures, unlike that of PAAs [7]
In this review, we summarize recent advances in the engineering of elongation factor-thermo unstable (EF-Tu), ribosomes, and tRNAs to improve the accommodation and peptidyl transfer of nPAAs that lead to an improvement in the incorporation efficiency of nPAAs

Summary

Introduction

Only the 20 proteinogenic amino acids (PAAs), the 19 L-α-amino acids, and glycine are available as building blocks for peptide/protein synthesis. Fujino et al reported in a 2013 paper that 19 D-α-amino acids were classified into three groups based on their single incorporation efficiency, and those classified in the top “efficient” group, such as D-Phe and D-Ala, could not be consecutively introduced into a model peptide [4,5,8] They successfully introduced 16 kinds of β-amino acids, but the most efficient members for single incorporation, such as β-homoglycine (β-hGly), L-β-homoalanine (L-β-hAla), L-β-homoglutamine (L-β-hGln), and L-β-homophenylglycine (L-β-hPhg), could not be consecutively introduced [4,5,8]. These results seemed to indicate a limitation of the translation system for the incorporation of such “exotic” amino acids. Solutions: a) Development of mutant EF-Tu that has higher binding affinity to D-/β-aminoacyl-tRNAs b) Engineering of T-stem of tRNA to improve EF-Tu binding c) Development of mutant ribosome compatible with D-/β-amino acids d) Introduction of a D-arm motif of tRNA that recruits EF-P onto ribosome

EF-Tu Engineering to Compensate for Poor Affinity to nPAA-tRNAs

Ribosome Engineering to Improve Single Incorporation of Exotic Amino Acids

Perspectives