The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme

Structures of Methanomethylophilus alvus Pyrrolysine tRNA-Synthetases Support the Need for De Novo Selections When Altering the Substrate Specificity

“…The diversity of natural PylRS orthologs provides a large inventory of potential GCE platforms that are, or can easily be made, orthogonal to each other. , Yet, at the same time, this diversity complicates direct transfer of PylRS functionality and evolvability from one PylRS to another. In our accompanying article, as well in work by others, , this complication became evident by our inability to make robust Ma PylRSs using prior knowledge of the Mb PylRS specificity and the AST strategy. To understand the origins of this failure, we took advantage of the increased stability and solubility of the Ma PylRS and report here crystal structures of Ma PylRS variants with Acd and ATP bound that shed light on why a Ma Acd-AST derived from a highly functional Mb PylRS variant was not as efficient as a de novo selected Ma Acd-RS1 variant.…”

“…Although the sequence identity of the Ma PylRS and Mm/Mb PylRS catalytic domains is low (∼35%), the tertiary structures of the Mm catalytic domain and Ma PylRS are very similar (Cα RMSD = 1.2 Å) (Figure C), and the substrate recognition elements in the amino acid binding pocket appear well conserved (Figure D) . Consequently, it has been proposed that specificity for a particular ncAA can be “transplanted” from Mm/Mb PylRS variants to the Ma PylRS by re-creating the same constellation of active site mutations. , This “active site transplant” (AST) method has been used successfully to create Ma PylRS variants for incorporating lysine-like derivatives with bulky, hydrophobic head groups by transplanting up to two mutations rationally designed to enlarge the back end of the ncAA binding pocket (e.g., Y126A/Y206F). ,, Similarly, mutations designed to open space in the front end of the Mm/Mb PylRSs amino acid binding pocket (e.g., N166S or N166A/C168A) were sufficient to alter the Ma PylRS substrate selectivity for singly substituted phenylalanine derivatives. ,, However, attempts to transplant the five Mm PylRS active site mutations that confer specificity for acetyl-lysine into Ma PylRS were not successful . These observations suggest that the AST method can be effective for creating Ma PylRS variants where only one or two mutations are required, but more extensively remodeled active sites may not be readily transferred across the platforms.…”

“…2,6,7 However, attempts to transplant the five Mm PylRS active site mutations that confer specificity for acetyllysine into Ma PylRS were not successful. 8 These observations suggest that the AST method can be effective for creating Ma PylRS variants where only one or two mutations are required, but more extensively remodeled active sites may not be readily transferred across the platforms.…”

Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids

“…The most widely used GCE platforms are derived from the closely related Methanosarcina barkeri ( Mb ) and Methanosarcina mazei ( Mm ) pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs, in part because they are specified by the amber codon UAG, they are orthogonal in both prokaryotic and eukaryotic translational systems, and they have amino acid-binding pockets that can be evolved to accept chemically diverse ncAAs . Shortcomings of the Mb and Mm PylRS pairs are that they frequently express as insoluble proteins, they are susceptible to proteolysis, they tend to localize to the nucleus, and they are of limited utility in yeast expression hosts. − These challenges can limit the efficiency of in vivo ncAA incorporation and the utility of the platforms for cell-free protein synthesis and kinetic characterization. More Pyl-based GCE platforms that overcome these issues would expand our capacity to incorporate multiple, structurally diverse ncAAs into target proteins.…”

“…This might be particularly evident when Mm / Mb RS specificity for an ncAA is conferred by a constellation of interactions between the binding pocket and the ncAA made possible by a considerable restructuring of the Mm active site. Indeed, recent attempts to transplant five mutations from an Mm active site specific for acetyl-lysine failed to yield a corresponding functional Ma variant perhaps because important interactions between the hydrophilic head groups and the active site pocket that define specificity were not satisfied in the architecture of the Ma active site.…”