Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion

Abstract

Over 20 years ago, the pyrrolysine encoding translation system was discovered in specific archaea. Our Review provides an overview of how the once obscure pyrrolysyl-tRNA synthetase (PylRS) tRNA pair, originally responsible for accurately translating enzymes crucial in methanogenic metabolic pathways, laid the foundation for the burgeoning field of genetic code expansion. Our primary focus is the discussion of how to successfully engineer the PylRS to recognize new substrates and exhibit higher in vivo activity. We have compiled a comprehensive list of ncAAs incorporable with the PylRS system. Additionally, we also summarize recent successful applications of the PylRS system in creating innovative therapeutic solutions, such as new antibody-drug conjugates, advancements in vaccine modalities, and the potential production of new antimicrobials.

Figure 1

Mechanism of in-frame amber stop-codon readthrough. 1) Activation of a ncAA by PylRS. 2) Binding and aminoacylation (“loading” or “charging”) of tRNA^Pyl by PylRS. 3) Aminoacylated tRNA^Pyl dissociates from PylRS. 4) Binding of tRNA^Pyl to elongation factor. 5) Binding to the aminoacyl site (A site) of the ribosome followed by movement to the peptidyl site (P site) for elongating the nascent polypeptide chain (NPC). Subsequently, tRNA^Pyl moves to the exit site (E site) and leaves the ribosome. 6) Translation terminates upon the arrival of a nonamber stop-codon at the A site. At that point, the release factor (RF) binds to the ribosome and initiates release of the polypeptide chain by dissolution of the translation complex.

Figure 2

A) Schematic representation of the three PylRS classes. NTD = N-terminal tRNA-binding domain; CTD = catalytic domain. B) Cartoons illustrating the tRNA^Pyl recognition mechanism of M. barkeri PylRS with the N-terminal fused small metal binding protein (SmbP = light pink) domain to enhance in-cell solubility. The 3D model structure (cartoon representation) was calculated using ColabFold (C-terminal domain = purple, N-terminal domain = light blue). The model alignment involved the N- and C-terminal domains, aligning them with the corresponding domains of Methanosarcina mazei (M. mazei) (PDB ID 5UD5) and D. hafniense (PDB ID 2ZNI) bound to tRNA^Pyl.^, The conserved active site residues are shown as sticks (cyan). For clarity and because it is unstructured, the linker region has been omitted. The N- and C-terminal domains of PylRS recognize the tRNA^Pyl in the anticodon stem region. Unlike canonical tRNAs, tRNA^Pyl has only a small variable arm, explaining its orthogonality to all other canonical tRNA/aaRS pairs. Additionally, the anticodon is not involved in the recognition mechanism. C, D, and E) Comparative analysis of E. coli tRNA^Ala and two tRNA^Pyl variants.^, For the illustration, E. coli tRNA^Ala was selected as a representative example of a canonical tRNA. The folding predictions were conducted using Geneious (version 7.1.9) employing the ViennaRNA Package. The color code indicates the predicted binding strength.

Figure 3

Aminoacylation reaction of tRNA. This reaction cascade is catalyzed by an aaRS. Driving force of the reaction is the pyrophosphate (PPi) hydrolysis. Hydrolysis of PPi is not depicted. 1) Activation of the amino acid. 2) Transfer of the amino acid to the tRNA. 3) Overall chemical reaction. Nucleophilic attack at the α position of ATP displaces PPi and transfers adenylate (5′-AMP) to AA forming aminoacyl-AMP (the reaction is called adenylation in nonribosomal peptide synthesis). The PPi is further hydrolyzed by inorganic pyrophosphatase to two Pi (−20 kJ/mol). This energy release constitutes the driving force of the reaction.

Figure 4

Natural and engineered Pyl-biosynthesis pathways. The natural Pyl biosynthesis^, was repurposed by establishing a new Pyl route via engineered PylC. The potential routes for incorporating ncAAs are highlighted in red, using existing biological synthetic pathways^, in combination with engineered PylRS variants. The metabolic pathways outlined in red, along with the specific PylRS variants facilitating the incorporation of these ncAAs, have been documented in the literature. The missing link is establishing the connection and optimizing these components to create a complete functional GCE pathway.

Figure 5

Group 1 of Lys derivatives that can be incorporated in vivo using the PylRS OTS. All ncAAs are Lys-derivatives containing a carbamate group starting at the Lys N^ε. Most of these ncAAs can be incorporated with the wild-type or PylRS(Y306A:Y384F, M. mazei notation) variant. Photo- or chemically caged post-translational modifications (blue), ncAAs containing bioorthogonal groups and are possible targets for site-specific bioconjugation (red), photo- or chemically caged ncAAs or cAAs (magenta), ncAAs containing functional groups which are useful for spectroscopic applications (orange), photo- or proximity triggered cross-linking ncAAs (violet), fluorescent ncAAs (cyan).

Figure 6

Group 2 of Lys derivatives that can be incorporated in vivo using the PylRS OTS. All ncAAs are Lys-derivatives with a Lys N^ε amide, or one with and ester (146). 140 and 141 are exceptions due to space constrains. Caged or noncaged post-translational modifications (blue), ncAAs containing bioorthogonal groups and are possible targets for site-specific bioconjugation (red), photo- or proximity triggered cross-linkable ncAAs (violet).

Figure 7

Group 3 contains short and bulky or larger bulky non-Lys ncAAs that can be incorporated in vivo using the PylRS OTS. Post-translational modifications (blue), ncAAs containing bioorthogonal groups and are possible targets for site-specific bioconjugation (red), photocaged ncAAs or cAAs (magenta), ncAAs containing functional groups which are useful for spectroscopic applications (orange), photo- or proximity triggered cross-linkable ncAAs (violet), fluorescent ncAAs (cyan).

Figure 8

Group 4 contains small and bulky His analogs, small aliphatic and small caged ncAAs. This group also contains the unusual α- and β-hydroxy acids, and all of them can be incorporated in vivo using the PylRS OTS. Some of the Lys derivatives contain ester which are not that common in the GCE field. Also, the in vivo incorporable α-hydroxy acids are very unusual substrates. They especially highlight the unique substrate promiscuity of the PylRS OTS. Post-translational modifications (blue), ncAAs containing bioorthogonal groups and are possible targets for site-specific bioconjugation (red), photocaged ncAAs or cAAs (magenta), ncAAs containing functional groups which are useful for spectroscopic applications (orange), photo- or proximity triggered cross-linkable ncAAs (violet), fluorescent ncAAs (cyan).

Figure 9

A) Nanobody containing 229, with a cell-penetrating peptide (CPP) tail, targeting PD-L1. B) Schematic comparison of the binding mechanism for conventional a nanobody and a covalently binding nanobody containing FSY(229), both targeting the HER2 receptor. Adapted from refs (226, 227). Copyright 2021, 2023 American Chemical Society.

Figure 10

Depiction of the site-specific conjugation of antibodies with radioisotopes for diagnostics and/or therapeutic applications.

Figure 11

Schematic depiction of epitope-directed antibody elicitation in mice with AcrK (108) and Kcr (109).

Figure 12

Workflow for pharmacological testing of tetherable heterobifunctional ligands for GPCRs containing a ncAA, adapted from ref (284). Copyright 2023 American Chemical Society.