Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme

Abstract

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Metadata

Item Type:	Article
Authors/Creators:	Dulchavsky, M. https://orcid.org/0000-0002-5253-8924 Mitra, R. Wu, K. Li, J. Boer, K. Liu, X. Zhang, Z. Vasquez, C. Clark, C.T. https://orcid.org/0000-0002-1267-1309 Funckes, K. Shankar, K. Bonnet-Zahedi, S. Siddiq, M. https://orcid.org/0000-0002-9186-1978 Sepulveda, Y. Suhandynata, R.T. Momper, J.D. Calabrese, A.N. https://orcid.org/0000-0003-2437-7761 George, O. https://orcid.org/0000-0002-3700-5003 Stull, F. https://orcid.org/0000-0002-2969-4224 Bardwell, J.C.A. https://orcid.org/0000-0003-1683-1944
Copyright, Publisher and Additional Information:	© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.
Keywords:	Biocatalysis; Biologics; Biophysics; Enzyme mechanisms; Structural biology
Dates:	Published: November 2023 Published (online): 28 September 2023 Accepted: 23 August 2023
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)
Depositing User:	Symplectic Publications
Date Deposited:	05 Jan 2024 15:11
Last Modified:	05 Jan 2024 15:11
Published Version:	http://dx.doi.org/10.1038/s41589-023-01426-y
Status:	Published
Publisher:	Springer Science and Business Media LLC
Identification Number:	10.1038/s41589-023-01426-y
Related URLs:	PubMed URL
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:205097

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Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme

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