An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

Abstract

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.

Keywords: Genetic code expansion; Noncanonical amino acid; PedH; Protein main-chain modification; Pyrrolysyl-tRNA synthetase.

Fig. 1

NcAA incorporation efficiency comparison between MmPylRS (N346A/C348A) and MbPylRS (N311A/C313A). A The chemical structures of 12 ncAAs used for testing incorporation efficiency. 1, 3-iodo-L-Phe (3-I-Phe); 2, 3-bromo-L-Phe (3-Br-Phe); 3, 3-methyl-L-Phe (3-Me-Phe); 4, 3-chloro-L-Phe (3-Cl-Phe); 5, 2-trifluoromethyl-L-Phe (2-CF3-Phe); 6, 3-nitro-L-Phe; 7, O-benzyl-L-tyrosine; 8, O-tert-butyl-L-tyrosine; 9, O-methyl-L-tyrosine; 10, 2-methyl-L-Phe (2-Me-Phe); 11, 2-bromo-L-Phe (2-Br-Phe); 12, 2-chloro-L-Phe (2-Cl-Phe). B Activity of Mm-NA/CA and Mb-NA/CA toward different ncAAs. Translation of the sfGFP reporter (UAG codon at position 2) by the two PylRS variants was measured by fluorescence intensity

Fig. 2

Incorporation efficiency of PylRS mutants toward 8 ncAAs during greedy evolution. A Incorporation efficiency of single mutants. Avg refers to the mutants’ average incorporation efficiency for the 8 ncAAs. B Incorporation efficiency of double mutants starting from T56P. C Incorporation efficiency of triple mutants starting from V31I/T56P. D Incorporation efficiency of quadruple mutants starting from V31I/T56P/A100E. E Comparison of incorporation efficiency between the optimal mutant obtained from each round of greedy evolution and IPYE, EIVLRT and Mb-NA/CA

Fig. 3

Analysis of MbPylRS complex structure bound with tRNA^Pyl. A Superposition of the MbPylRS-tRNA^Pyl complex predicted by AlphaFold2. B N-terminal residues selected for greedy evolution are represented as sticks, with V31, T56 and A100 represented as spheres. The secondary structure features of tRNA^Pyl are colored: blue, D loop; brown, anticodon stem; purple, anticodon; yellow, variable loop; orange, T loop; green, acceptor stem. C Close-up view of T56 and the surrounding residues. D Close-up view of V31 and the surrounding residues

Fig. 4

Molecular structures of the 16 novel ncAAs in four classes accepted by IPE variant and the incorporation efficiency of Mb-NA/CA and IPE toward these ncAAs. Structures of A halogenated Phes, B thienyl-L-alanines, C non-phenyl-substituted Phes, D para-substituted Phes. Incorporation efficiency of Mb-NA/CA and IPE toward E halogenated Phes, F thienyl-L-alanines, G non-phenyl-substituted Phes, H para-substituted Phes. 13, L-2,5-dichloroPhe; 14, L-2,4-difluoroPhe; 15, L-2,3-difluoroPhe; 16, L-2,5-difluoroPhe; 17, L-2,4,5-trifluoroPhe; 18, L-2,3,6-trifluoroPhe; 19, L-2,3-dichloroPhe; 20, 5-bromo-2-chloro-L-Phe; 21, L-2-(5-bromothienyl)alanine; 22, L-3-benzothienylalanine; 23, 3-(3-thienyl)-L-alanine; 24, 3-(2-thienyl)-L-alanine; 25, Phenyl-lactic acid; 26, 2-amino-2-phenylpropionic acid; 27, L-homoPhe; 28, 4-nitro-L-Phe

Fig. 5

Structural features of the 23/24-IPE-binding sites and the key residues. A Superposition of the binding conformations of 23 and 24 with the IPE-tRNA^Pyl monomer. 3-(3-thienyl)-L-alanine (23) is represented as cyan sticks, and 3-(2-thienyl)-L-alanine (24) is represented as salmon sticks. B Interaction between 24 and the surrounding residues. C Interaction between 23 and the surrounding residues

Fig. 6

Incorporation of 8 ncAAs into the 412 site of PedH. A Crystal structure of PedH (PDB ID 6ZCW) and close-up view around the 412 site of PedH. The light blue channel indicates the substrate channel predicted by Caver 3. B Structures of the 8 ncAAs incorporated into PedH. C Specific activities of the variants toward ethanol and methanol. D Specific activities of the variants toward (S)-2-butanol and (R)-2-butanol