Windle, CL, Simmons, KJ orcid.org/0000-0003-4846-9097, Ault, JR orcid.org/0000-0002-5131-438X et al. (4 more authors) (2017) Extending enzyme molecular recognition with an expanded amino acid alphabet. Proceedings of the National Academy of Sciences, 114 (10). pp. 2610-2615. ISSN 1091-6490
Abstract
Natural enzymes are constructed from the twenty proteogenic amino acids, which may then require post-translational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of non-canonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different non-canonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically-modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modelling studies revealed that the unique shape and functionality of the non-canonical side chain enabled the active site to be remodelled to enable more efficient stabilisation of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | This is an author produced version of a journal article published in Proceedings of the National Academy of Sciences. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Protein engineering; aldolases; chemical modification |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Synthetic Biology (Leed) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/M012573/1 BBSRC BB/N002091/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 26 Jan 2017 12:13 |
Last Modified: | 06 Oct 2020 10:42 |
Published Version: | https://doi.org/10.1073/pnas.1616816114 |
Status: | Published |
Publisher: | National Academy of Sciences |
Identification Number: | 10.1073/pnas.1616816114 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:111212 |