Exploring higher plant Rubisco activase function

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-d...

Full description

Saved in:
Bibliographic Details
Main Author: Ng, Jediael Zheng Ying
Other Authors: Oliver Mueller-Cajar
Format: Thesis-Doctor of Philosophy
Language:English
Published: Nanyang Technological University 2021
Subjects:
Online Access:https://hdl.handle.net/10356/151272
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-151272
record_format dspace
spelling sg-ntu-dr.10356-1512722023-02-28T18:46:57Z Exploring higher plant Rubisco activase function Ng, Jediael Zheng Ying Oliver Mueller-Cajar School of Biological Sciences cajar@ntu.edu.sg Science::Biological sciences::Biochemistry 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. Doctor of Philosophy 2021-06-23T11:52:39Z 2021-06-23T11:52:39Z 2021 Thesis-Doctor of Philosophy Ng, J. Z. Y. (2021). Exploring higher plant Rubisco activase function. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/151272 https://hdl.handle.net/10356/151272 10.32657/10356/151272 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences::Biochemistry
spellingShingle Science::Biological sciences::Biochemistry
Ng, Jediael Zheng Ying
Exploring higher plant Rubisco activase function
description 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.
author2 Oliver Mueller-Cajar
author_facet Oliver Mueller-Cajar
Ng, Jediael Zheng Ying
format Thesis-Doctor of Philosophy
author Ng, Jediael Zheng Ying
author_sort Ng, Jediael Zheng Ying
title Exploring higher plant Rubisco activase function
title_short Exploring higher plant Rubisco activase function
title_full Exploring higher plant Rubisco activase function
title_fullStr Exploring higher plant Rubisco activase function
title_full_unstemmed Exploring higher plant Rubisco activase function
title_sort exploring higher plant rubisco activase function
publisher Nanyang Technological University
publishDate 2021
url https://hdl.handle.net/10356/151272
_version_ 1759857267575881728