Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function

Abstract

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.

Keywords: Non-canonical amino acid; Phenylalanyl-tRNA synthetase; Pyrrolysyl-tRNA synthetase; Superfolder green fluorescent protein.

Figure 1

Relative fluorescence emission of sfGFP in twenty natural amino acids with FRS variants. The y-axis units are the percentages of fluorescence intensity from sfGFP with Ser2TAG mutation compared to the fluorescence intensity of wild type sfGFP. The fluorescence intensity was measured by excitation wavelength in 485 nm/20 nm and emission wavelength 528 nm/20 nm. Each fluorescence intensity was presented after subtraction with the value from the control experiment without adding the amino acid.

Figure 2

Mass spectrometric analysis of sfGFP-F2 that was produced using FRS3-tRNA^Pyl pair in M9 medium. The LC-MS/MS spectra of the MXKGEELF (X denotes F) fragment from the full-length GFP-F2 protein. b⁰ stands for b-H₂O.

Figure 3

Range of substrate specificity of FRS enzymes. Suppression of the sfGFP-UAG2 gene by the library of ncAA-tRNA^Pyl was measured by fluorescence intensity. Four wells (A1, A2, I1 and I2) were set as control experiments without adding any ncAA to detect the background signals. (A) The substrate specificity profile of FRS3. (B) The substrate specificity profile of FRS2. (C) The substrate specificity profile of FRS1 The ncAA chemical structures are given in Scheme S1 and Table S2.

Figure 4

The comparison of FRS3, FRS1, PylRS and E. coli PheRS structures. Structural models of FRS3 and FRS1 with Phe were generated by geometry-minimization using PHENIX [20] based on the PylRS and PylRS-derived O-methyl-tyrosyl-tRNA synthetase structures (PDB ID: 2ZIM [10] and 3QTC [12], respectively). The gray surface model indicated the volume and shape of the substrate-binding pocket; the Phe substrate (in magenta) and residues surrounding the substrate-binding pocket are shown as sticks. The residues that were chosen for the library are underlined. All figures were prepared using Pymol software (the PyMOL Molecular Graphics System, Schrödinger, LLC). (A) FRS3: Ala302Phe and Cys348Phe are located close to the substrate Phe (red sticks). (B) The co-crystal structure of E. coli PheRS with Phe (PDB ID: 3PCO [11]). Phe248 and Phe250 are conserved in bacterial PheRSs and contribute to the substrate recognition by π-interactions (red sticks). (C) FRS1: The arrangement of Ala302 and Asn346Ala can form a larger pocket for Phe analogs. (D) The co-crystal structure of PylRS with Pyl-AMP.