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Structural and functional analysis of GUN4 and ChlH subunits of the magnesium chelatase enzyme

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posted on 28.03.2022, 13:26 by Shabnam Tarahi Tabrizi
In plants and algae the Genomes Uncoupled 4 (GUN4) is a nuclear-encoded, chloroplast localized, porphyrin binding protein implicated in retrograde signaling between the chloroplast and nucleus. Functionally, it enhances Mg-chelatase activity, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway, possibly through the removal of Mg protoporphyrin IX (Mg-PPIX) the product of the reaction. Our in vitro results show that purified GUN4 increases the production of PPIX-generated singlet oxygen in the light by a factor of five compared with PPIX alone. Additionally the functional GUN4-PPIX-ChlH complex and GUN4-Mg-PPIX generate singlet oxygen at a reduced rate compared with GUN4-PPIX. Recent evidence points to singlet oxygen being a possible plastid to nucleus signal, possibly through second messengers. The light dependent singlet oxygen generation by GUN4-PPIX may be part of a signal transduction pathway from the chloroplast to the nucleus which senses the availability and flux of PPIX through the chlorophyll biosynthetic pathway in order to control the concentrations of enzymes and chlorophyll binding proteins in the chloroplast. Additionally, unlike the cyanobacterial GUN4, the chloroplastic orthologues have an extra C-terminal domain that is phosphorylated and is required for magnesium chelatase activity. The low resolution solution structure of full length GUN4 at ~20 Å was determined, by using small-angle X-ray scattering (SAXS) and report that the protein has a more elongated structure compared to the cyanobacterial protein. Also, the first crystal structure of truncated eukaryotic GUN4 from Chlamydomonas reinhardtii was solved. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to our understanding of how GUN4 binds PPIX or Mg-PPIX.


Table of Contents

Chapter 1. Introduction -- Chapter 2. Materials and methods -- Chapter 3. Structure characterization of GUN4, ChlH and GUN4-ChlH-PPIX complex by Small Angle X-ray Scattering (SAXS) -- Chapter 4. Crystallization and structure determination of GUN4 from Chlamydomonas reinhardtii -- Chapter 5. GUN4 of Chlamydomonas reinhardtii enhances singlet oxygen production in the presence of light and protoporphyrin IX (Publication III: prepared for submission) -- Chapter 6. General discussion.


Empirical thesis. Bibliography: pages 163-174

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


PhD, Macquarie University, Faculty of Science and Engineering, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award


Principal Supervisor

Robert Willows


Copyright Shabnam Tarahi Tabrizi 2016. Copyright disclaimer: http://www.copyright.mq.edu.au




1 online resource (217 pages) colour illustrations

Former Identifiers

mq:46379 http://hdl.handle.net/1959.14/1084514