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Preprint
The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix
Research Square, Vol.23 February 2023
Research Square Company
2023
Abstract
A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenases, named after their unique metalloclusters containing either Mo-, V- or Fe, that convert N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK greatly improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase genes within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.
Details
- Title
- The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix
- Authors
- Ema Johnston (Author) - Commonwealth Scientific and Industrial Research OrganisationShoko Okada (Author) - Commonwealth Scientific and Industrial Research OrganisationChristina Gregg (Author) - Commonwealth Scientific and Industrial Research OrganisationAndrew Warden (Author) - Commonwealth Scientific and Industrial Research OrganisationVivien Rolland (Author) - Commonwealth Scientific and Industrial Research OrganisationVanessa Gillespie (Author) - Commonwealth Scientific and Industrial Research OrganisationKeren Byrne (Author) - Commonwealth Scientific and Industrial Research OrganisationMichelle Colgrave (Author) - Commonwealth Scientific and Industrial Research OrganisationAndy Eamens (Author) - University of the Sunshine Coast, Queensland, School of Health - BiomedicineRobert Silas Allen (Corresponding Author) - Commonwealth Scientific and Industrial Research OrganisationCraig Wood (Author) - Commonwealth Scientific and Industrial Research Organisation
- Publication details
- Research Square, Vol.23 February 2023
- Publisher
- Research Square Company
- Date published
- 2023
- DOI
- 10.21203/rs.3.rs-2567372/v1
- ISSN
- 2693-5015
- Copyright note
- This work is licensed under a CC BY 4.0 License.
- Organisation Unit
- School of Health - Biomedicine
- Language
- English
- Record Identifier
- 99719198402621
- Output Type
- Preprint
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