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|Title:||Exploring higher plant Rubisco activase function||Authors:||Ng, Jediael Zheng Ying||Keywords:||Science::Biological sciences::Biochemistry||Issue Date:||2021||Publisher:||Nanyang Technological University||Source:||Ng, J. Z. Y. (2021). Exploring higher plant Rubisco activase function. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/151272||Abstract:||The key photosynthetic enzyme Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inhibition by tightly bound sugar phosphates. Consequently, it is reliant on the essential AAA+ chaperone Rubisco activase (Rca) to relieve active site inhibition. While the ability to carry out ATP-driven conformational remodelling of substrates is intrinsic to all activases, distinct mechanisms are utilized by different activase classes. This is vitally pertinent for higher plant activases, whose mechanism remains poorly understood despite its potential as a crop improvement target. Biochemical and structural data implicates the hexamer as the functional unit of the plant type activase, with the convex surface of Rca established as the Rubisco interacting face. To assess the contribution of individual residues for activase function in the context of a functional hexamer, subunit mutant doping experiments were conducted using a suite of activase mutants with impaired Rca activity. Rubisco interacting residues at the periphery of the disc were observed to be less critical for activase function compared to residues located in proximity to the central pore, hinting at the importance of the central pore to the mechanism of Rubisco activation in plants. Leveraging on recent advances in recombinant expression of higher plant Rubisco, similar structure guided mutagenesis of residues on the surface of Rubisco were then carried out to identify Rca interacting elements on Rubisco. Rca activity was found to be sensitive to both truncations and mutations at the conserved flexible N-terminus of the Rubisco large subunit. Coupled together, our results are consistent with a model where Rca remodels Rubisco by transient threading of the Rubisco large subunit N-terminus through its central pore. Finally, to explore further possible avenues for crop enhancement, a Rubisco activase-like isoform that is conserved amongst all higher plants is also investigated. Despite negligible reactivation rates when assayed with inhibited Rubisco complexes, the activase-like isoform was observed to interact with both canonical activases and Rubisco complexes, thus suggesting of a currently uncharacterized role within the plant Rubisco-Rubisco activase system.||URI:||https://hdl.handle.net/10356/151272||DOI:||10.32657/10356/151272||Rights:||This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).||Fulltext Permission:||embargo_20220609||Fulltext Availability:||With Fulltext|
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