Pyrrolysyl-tRNA synthetase, an aminoacyl-tRNA synthetase for genetic code expansion

Abstract

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22^nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNA^Pyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNA^Pyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.

Keywords: genetic code expansion; non-canonical amino acid; pyrrolysyl-tRNA synthetase; stop codon suppression; synthetic biology; tRNAPyl.

Figure 1

Schematic representation of stop codon suppression strategy. PylRS aminoacylates an orthogonal suppressor tRNA (anticodon CUA) with a non-cognate amino acid (ncAA). NcAA-tRNA^Pyl_CUA is delivered to the ribosome by the host elongation factor EF-Tu. At the ribosome, the ncAA-tRNA^Pyl_CUA decodes an internal stop codon (UAG) and the ncAA becomes incorporated in the protein of interest.

Figure 2

Structure of the PylRS•tRNA^Pyl complex from D. hafniense (PDB ID 2ZNI) from two perspectives. Individual monomers of the PylRS are shown in grey and magenta, tRNA(I) in light blue and tRNA(II) pink.

Figure 3

PylRS topology and α-amino group recognition in the active site. The enzyme is composed of two identical monomers (grey and magenta) forming a homodimer. Characteristic class II aaRS motifs are accentuated: motif 1 that forms part of the dimer interface is shown in green and motif 2 loop (involved in tRNA 3′-CCA recognition) in blue. Pyl is bound in the active site and shown in yellow. (Inset) Idiosyncratic recognition of the Pyl α-amino group in M. mazei PylRS (PDB ID 2ZCE). Instead of directly recognizing the α-amino group of the AA substrate PylRS uses a water molecule bound to the Asn-346. Water molecule (cyan) is shown as a ball. Hydrogen bonds are shown as black dashed lines (B) Malleability of the active site as illustrated by comparison of the wild-type enzyme and a norbornene charging mutant (PDB ID 2Q7H and 4BWA, respectively). The wild-type enzyme is shown in magenta and the triple Y306G/Y384F/I405R mutant in cyan. Pyl-AMP and norbornene-AMP (nonhydrolyzable analogs) are given as stick representation in yellow and in green, respectively, with transparent surfaces indicating the substrate-binding pocket. In the triple mutant the Y306G mutation at the base of the AA binding pocket enlarges the binding site thus allowing placement of the norbornene head group (this space is normally occupied by Tyr-306 side chain, see wild-type structure). In the wild-type enzyme Asn-346 forms a hydrogen bond with the carbonyl-amide linkage of Pyl side chain. In contrast, the carbamate linkage of the norbornene does not form a bond with Asn-346 but with Cys-348.

Figure 4

NcAAs that can be co-translationally incorporated into proteins for bioorthogonal labelling through Cu(I) catalyzed cycloaddition (N^ε-((prop-2-yn-1-yloxy)carbonyl)-l-lysine (1), N^ε-((2-azidoethoxy)carbonyl)-l-lysine (2), N^ε-((2R,3S)-3-ethynyltetrahydrofuran-2-carbonyl)-l-lysine (3), N^ε-((S)-3-aminohex-5-ynoyl)-l-lysine (4)) or through inverse-electron-demand Diels–Alder reaction (N^ε-((((R,E)-cyclooct-4-en-1-yl)oxy)carbonyl)-l-lysine (5), N^ε-((cyclooct-2-yn-1-yloxy)carbonyl)-l-lysine (6), N^ε-((((1R,8S)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-l-lysine (7), N^ε-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)-l-lysine (8)).

Figure 5

NcAAs with photo-reactive substituents for photo-crosslinking (N^ε-((3-(3-methyl-3H-diazirin-3-yl)propyl)carbamoyl)-l-lysine (9)) and photo-caged ncAAs (N^ε-(tert-butoxycarbonyl)-l-lysine (10), N^ε-(tert-butoxycarbonyl)-N^ε-methyl-l-lysine (11), N^ε-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-l-lysine (12), N^ε-methyl-N^ε-(((2-nitrobenzyl)oxy)carbonyl)-l-lysine (13), N^ε-((benzyloxy)carbonyl)-N^ε-methyl-l-lysine (14), N^ε-((allyloxy)carbonyl)-N^ε-methyl-l-lysine (15)). NcAAs for chemical ligation purposes (2S)-2,6-diamino-5-mercaptohexanoic acid (16). NcAAs that can be synthesized in E. coli using the pylBCD biosynthetic cluster: N^ε-((R)-3,4-dihydro-2H-pyrrole-2-carbonyl)-l-lysine (17), N^ε-((R)-3,4-dihydro-2H-pyrrole-2-carbonyl)-l-lysine (18) N^ε-((2R,3S)-3-ethynyl-3,4-dihydro-2H-pyrrole-2-carbonyl)-l-lysine (19).