Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

Mapping Intimacies ◽

10.21203/rs.3.rs-468406/v2 ◽

2021 ◽

Author(s):

Thomas Williams ◽

Yu-Hsuan Tsai ◽

Louis Luk

Keyword(s):

Genetic Code ◽

Secondary Amine ◽

Transfer Hydrogenation ◽

Protein Scaffolds ◽

Artificial Enzyme ◽

Enzyme Design ◽

Catalytically Active ◽

Amine Catalysis ◽

Genetic Code Expansion ◽

Distinctive Role

Abstract Here, incorporation of secondary amine by genetic code expansion was used to expand the potential protein templates for artificial enzyme design. Pyrrolysine analogue containing a D-proline could be stably incorporated into proteins, including the multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Both modified scaffolds were catalytically active, mediating transfer hydrogenation with a relaxed substrate scope. The protein templates played a distinctive role in that, while the LmrR variants were confined to the biomimetic BNAH as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for reactions. Due to the cofactor compatibility, the DHFR secondary amine catalysis could also be coupled to an enzymatic recycling scheme. This work has illustrated the unique advantages of using proteins as hosts, and thus the presented concept is expected to find uses in enabling tailored secondary amine catalysis.

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NADPH-dependent Secondary Amine Organocatalysis hosted by a Nucleotide-binding Domain

10.21203/rs.3.rs-468406/v1 ◽

2021 ◽

Author(s):

Thomas Williams ◽

Yu-Hsuan Tsai ◽

Louis Luk

Keyword(s):

Secondary Amine ◽

Nucleotide Binding ◽

Unnatural Amino Acid ◽

Binding Domain ◽

Transfer Hydrogenation ◽

Protein Scaffolds ◽

Artificial Enzyme ◽

Hybrid Catalyst ◽

Nucleotide Binding Domain ◽

Nadph Regeneration

Abstract The concept of organocatalysis has been applied to facilitate “new-to-nature” reaction modes via artificial enzyme design. However, it remains challenging to recruit structurally complex natural molecules as synthetic reagents. Here, we have reported a generic design strategy that allows generation of a NADPH-dependent hybrid catalyst whose action is orchestrated by a secondary amine; this system recruits a reaction mode not commonly seen among enzymes, whilst involving an intricate cofactor that cannot be used by existing organocatalysts. A secondary amine organocatalytic motif was incorporated into protein scaffolds as an unnatural amino acid by expansion of the genetic code. When introduced into the multidrug binding protein LmrR, a hybrid catalyst accepting α,β-unsaturated carbonyl substrates for transfer hydrogenation was established but was confined to the much-simplified biomimetic benzyl dihydronicotinamide (BNAH). Conversely, dihydrofolate reductase (DHFR) contains a nucleotide binding domain and can be converted into a hybrid catalyst that favourably uses NADPH for reaction, thus highlighting the importance of choosing an appropriate scaffold. The DHFR-hosted system tolerates a range of aldehyde substrates and can be coupled with an enzymatic NADPH regeneration scheme. The presented engineering approach can be readily extended to other protein scaffolds for use of different natural molecules in non-natural reaction modes.

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Faculty Opinions recommendation of A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1057894.512855 ◽

2007 ◽

Author(s):

Niles Lehman

Keyword(s):

Genetic Code ◽

Genetic Encoding ◽

Genetic Code Expansion

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Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

SSRN Electronic Journal ◽

10.2139/ssrn.3244022 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuo Yanagisawa ◽

Mitsuo Kuratani ◽

Eiko Seki ◽

Nobumasa Hino ◽

Kensaku Sakamoto ◽

...

Keyword(s):

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Genetic Code Expansion

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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 ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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