Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Mutations on Substrate Specificity

Abigail V Sharrock
,
Jeff S Mumm
,
Gintautas Bagdžiūnas
ORCID logo ,
Narimantas Čėnas
,
Vickery L Arcus
* ,
David F. Ackerley
* ORCID logo
Version 1 : Received: 9 March 2023 / Approved: 17 March 2023 / Online: 17 March 2023 (03:22:59 CET)

How to cite: Sharrock, A.V.; Mumm, J.S.; Bagdžiūnas, G.; Čėnas, N.; Arcus, V.L.; Ackerley, D.F. The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Mutations on Substrate Specificity. Preprints 2023, 2023030315. https://doi.org/10.20944/preprints202303.0315.v1. Sharrock, A.V.; Mumm, J.S.; Bagdžiūnas, G.; Čėnas, N.; Arcus, V.L.; Ackerley, D.F. The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Mutations on Substrate Specificity. Preprints 2023, 2023030315. https://doi.org/10.20944/preprints202303.0315.v1.

Abstract

Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase (“NTR 2.0”) for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.

Keywords

Type I nitroreductase; NfsB; Crystal structure; Prodrug; Metronidazole; Targeted cellular ablation; Tinidazole; CB1954; Dinitrotoluene

Subject

LIFE SCIENCES, Biochemistry

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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