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

Pyrrolysyl-tRNA synthetase / Genetic code expansion / Noncanonical amino acid / Protein main-chain modification / PedH

References

Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 2015, 1–2: 19-25.

Brittain WDG, Lloyd CM, Cobb SL. Synthesis of complex unnatural fluorine-containing amino acids. J Fluor Chem, 2020, 239: 109630.

Brown W, Liu J, Deiters A. Genetic code expansion in animals. ACS Chem Biol, 2018, 13(9): 2375-2386.

Bryson DI, Fan C, Guo LT, Miller C, Soll D, Liu DR. Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol, 2017, 13(12): 1253-1260.

Cobb SL, Murphy CD. ¹⁹F NMR applications in chemical biology. J Fluorine Chem, 2009, 130(2): 132-143.

Cui Y, Chen Y, Liu X, Dong S, Ye T, Qiao Y, Mitra R, Han J, Li C, Han X, Liu W, Chen Q, Wei W, Wang X, Du W, Tang S, Xiang H, Liu H, Liang Y, Houk KN, Wu B. Computational redesign of a PETase for plastic biodegradation under ambient condition by the GRAPE strategy. ACS Catal, 2021, 11(3): 1340-1350.

Englert M, Nakamura A, Wang YS, Eiler D, Soll D, Guo LT. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog. Nucleic Acids Res, 2015, 43(22): 11061-11067.

Fuhrer TJ, Houck M, Corley CA, Iacono ST. Theoretical explanation of reaction site selectivity in the addition of a phenoxy group to perfluoropyridine. J Phys Chem A, 2019, 123(44): 9450-9455.

Good NM, Vu HN, Suriano CJ, Subuyuj GA, Skovran E, Martinez-Gomez NC. Pyrroloquinoline quinone ethanol dehydrogenase in Methylobacterium extorquens AM1 extends lanthanide-dependent metabolism to multicarbon substrates. J Bacteriol, 2016, 198(22): 3109-3118.

Guo LT, Wang YS, Nakamura A, Eiler D, Kavran JM, Wong M, Kiessling LL, Steitz TA, O'Donoghue P, Soll D. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. P Natl Acad Sci USA, 2014, 111(47): 16724-16729.

Huang J, MacKerell AD Jr. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J Comput Chem, 2013, 34(25): 2135-2145.

Jahn B, Pol A, Lumpe H, Barends TRM, Dietl A, Hogendoorn C, Op den Camp HJM, Daumann LJ. Similar but not the same: first kinetic and structural analyses of a methanol dehydrogenase containing a europium ion in the active site. ChemBioChem, 2018, 19(11): 1147-1153.

Jahn B, Jonasson NSW, Hu H, Singer H, Pol A, Good NM, den Camp H, Martinez-Gomez NC, Daumann LJ. Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster’s blue in methanol dehydrogenase assays. J Biol Inorg Chem, 2020, 25(2): 199-212.

Jiang HK, Lee MN, Tsou JC, Chang KW, Tseng HW, Chen KP, Li YK, Wang YS. Linker and N-terminal domain engineering of pyrrolysyl-tRNA synthetase for substrate range shifting and activity enhancement. Front Bioeng Biotech, 2020

Kavran JM, Gundliapalli S, O'Donoghue P, Englert M, Soell D, Steitz TA. Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation. P Natl Acad Sci USA, 2007, 104(27): 11268-11273.

Keltjens JT, Pol A, Reimann J, Op den Camp HJ. PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference. Appl Microbiol Biotechnol, 2014, 98(14): 6163-6183.

Koch NG, Goettig P, Rappsilber J, Budisa N. Engineering Pyrrolysyl-tRNA synthetase for the incorporation of non-canonical amino acids with smaller side chains. Int J Mol Sci, 2021, 22(20): 11194.

Kurra Y, Odoi KA, Lee YJ, Yang Y, Lu T, Wheeler SE, Torres-Kolbus J, Deiters A, Liu WR. Two rapid catalyst-free click reactions for in vivo protein labeling of genetically encoded strained alkene/alkyne functionalities. Bioconjug Chem, 2014, 25(9): 1730-1738.

Li JC, Liu T, Wang Y, Mehta AP, Schultz PG. Enhancing protein stability with genetically encoded noncanonical amino acids. J Am Chem Soc, 2018, 140(47): 15997-16000.

Neumann H, Peak-Chew SY, Chin JW. Genetically encoding N ^ε-acetyllysine in recombinant proteins. Nat Chem Biol, 2008, 4(4): 232-234.

Owens AE, Grasso KT, Ziegler CA, Fasan R. Two-tier screening platform for directed evolution of aminoacyl-tRNA synthetases with enhanced stop codon suppression efficiency. Chem Bio Chem, 2017, 18(12): 1109-1116.

Schrodinger LLC. The PyMOL molecular graphics system. Version, 2015, 1: 8.

Shandell MA, Tan Z, Cornish VW. Genetic code expansion: a brief history and perspective. Biochemistry-Us, 2021, 60(46): 3455-3469.

Sharma V, Wang YS, Liu WR. Probing the catalytic charge-relay system in alanine racemase with genetically encoded histidine mimetics. ACS Chem Biol, 2016, 11(12): 3305-3309.

Sharma V, Zeng Y, Wang WW, Qiao Y, Kurra Y, Liu WR. Evolving the N-terminal domain of pyrrolysyl-tRNA synthetase for improved incorporation of noncanonical amino acids. Chem Bio Chem, 2018, 19(1): 26-30.

Spinck M, Piedrafita C, Robertson WE, Elliott TS, Cervettini D, de la Torre D, Chin JW. Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles. Nat Chem, 2023, 15(1): 61-69.

Suzuki T, Miller C, Guo LT, Ho JML, Bryson DI, Wang YS, Liu DR, Soll D. Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nat Chem Biol, 2017, 13(12): 1261-1266.

Tharp JM, Wang YS, Lee YJ, Yang Y, Liu WR. Genetic incorporation of seven ortho-substituted phenylalanine derivatives. ACS Chem Biol, 2014, 9(4): 884-890.

Tharp JM, Ehnbom A, Liu WR. tRNA^Pyl: Structure, function, and applications. RNA Biol, 2018, 15(4–5): 441-452.

Tuley A, Wang YS, Fang X, Kurra Y, Rezenom YH, Liu WR. The genetic incorporation of thirteen novel non-canonical amino acids. Chem Commun (camb), 2014, 50(20): 2673-2675.

Vatansever EC, Yang KS, Geng ZZ, Qiao Y, Li P, Xu S, Liu WR. A designed, highly efficient pyrrolysyl-tRNA synthetase mutant binds o-chlorophenylalanine using two halogen bonds. J Mol Biol, 2022, 434(8): 167534.

Wan W, Tharp JM. Liu WR (2014) pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Bba-Proteins Proteom, 1844, 6: 1059-1070.

Wang YS, Russell WK, Wang Z, Wan W, Dodd LE, Pai PJ, Russell DH, Liu WR. The de novo engineering of pyrrolysyl-tRNA synthetase for genetic incorporation of L-phenylalanine and its derivatives. Mol Biosyst, 2011, 7(3): 714-717.

Wang YS, Fang X, Wallace AL, Wu B, Liu WR. A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. J Am Chem Soc, 2012, 134(6): 2950-2953.

Wehrmann M, Billard P, Martin-Meriadec A, Zegeye A, Klebensberger J. Functional role of lanthanides in enzymatic activity and transcriptional regulation of pyrroloquinoline quinone-dependent alcohol dehydrogenases in Pseudomonas putida KT2440. Mbio, 2017, 8(3): e00570-e1517.

Wehrmann M, Elsayed EM, Köbbing S, Bendz L, Lepak A, Schwabe J, Wierckx N, Bange G, Klebensberger J. Engineered PQQ-dependent alcohol dehydrogenase for the oxidation of 5-(Hydroxymethyl)furoic acid. ACS Catal, 2020, 10(14): 7836-7842.

Wilkinson HC, Dalby PA. Fine-tuning the activity and stability of an evolved enzyme active-site through noncanonical amino-acids. Febs J, 2021, 288(6): 1935-1955.

Williams TL, Iskandar DJ, Nodling AR, Tan Y, Luk LYP, Tsai YH. Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency. Amino Acids, 2021, 53(1): 89-96.

Young DD, Schultz PG. Playing with the Molecules of Life. ACS Chem Biol, 2018, 13(4): 854-870.

Young DD, Young TS, Jahnz M, Ahmad I, Spraggon G, Schultz PG. An evolved aminoacyl-tRNA Synthetase with atypical polysubstrate specificity. Biochemistry-Us, 2011, 50(11): 1894-1900.

Yu W, He X, Vanommeslaeghe K, MacKerell AD Jr. Extension of the CHARMM general force field to sulfonyl-containing compounds and its utility in biomolecular simulations. J Comput Chem, 2012, 33(31): 2451-2468.

Yu H, Ma S, Li Y, Dalby PA. Hot spots-making directed evolution easier. Biotechnol Adv, 2022, 56: 107926.

Funding

National Natural Science Foundation of China(Grant No. 22108245); Key Research and Development Program of China(Grant No. 2022YFA0913000); Fundamental Research Funds for the Central Universities(Grant No. 226-2022-00214)