Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool

Abstract

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA(Pyl). Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA(Pyl). These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

Keywords: Amber codon; Genetic code expansion; Hydroxy acids; Non-canonical amino acids; Pyrrolysine.

Figure 1

The essential four components of the Pyl incorporation machinery: pyrrolysyl-tRNA synthetase, ${\text{tRNA}}_{\text{CUA}}^{\text{Pyl}}$, Pyl, and amber codon.

Figure 2

The biosynthetic pathway of Pyl.

Figure 3

(A) The structure of Thermus thermophilus PheRS with phenyl-AMP bound at the active site (PDB entry: 1B7Y). (B) The structure of M. mazei PylRS with pyrrolysyl-AMP bound at the active site (PDB entry: 2ZIM). (C) The overlay of the two structures.

Figure 4

(A) The active site of Pyl. (B) The contoured deep substrate binding cavity of PylRS. Pyl-AMP is shown in sticks.

Figure 5

(A) The recognition of the substrate α-amine at the active site of PylRS. Side chains of residues 300–302 are not shown for a clear view of the hydrogen bond network of α-amine. (B) The recognition of the substrate α-amine at the active site of T. thermophilus PheRS.

Figure 6

The D. hafniense PylRS complex with tRNA^Pyl. PylRS forms a homodimer that associates with two ${\text{tRNA}}_{\text{CUA}}^{\text{Pyl}}$ molecules. Three anticodon nucleotides in both ${\text{tRNA}}_{\text{CUA}}^{\text{Pyl}}$ molecules are shown in spheres and colored in magenta.

Figure 7

NCAAs that serve as substrates of the native PylRS.

Figure 8

Lysine derivatives that have been genetically incorporated into proteins using engineering PylRS mutants in coordination with tRNA^Pyl.

Figure 9

Phenylalanine derivatives that have been genetically incorporated into proteins using engineering PylRS mutants in coordination with tRNA^Pyl.

Figure 10

α-Hydroxy acids that have been genetically incorporated into proteins using engineering PylRS mutants in coordination with tRNA^Pyl.

Figure 11

A general platform of incorporating two different NCAAs into one protein for a catalyst-free and one-pot dual labeling.