Abstract
The Mg-dechelatase enzyme encoded by the Stay-Green (SGR) gene catalyzes Mg2+ dechelation from chlorophyll a. This reaction is the first committed step of chlorophyll degradation pathway in plants and is thus indispensable for the process of leaf senescence. There is no structural information available for this or its related enzymes. This study aims to provide insights into the structure and reaction mechanism of the enzyme through biochemical and computational analysis of an SGR homolog from the Chloroflexi Anaerolineae (AbSGR-h). Recombinant AbSGR-h with its intact sequence and those with mutations were overexpressed in Escherichia coli and their Mg-dechelatase activity were compared. Two aspartates – D34 and D62 were found to be essential for catalysis, while R26, Y28, T29 and D114 were responsible for structural maintenance. Gel filtration analysis of the recombinant AbSGR-h indicates that it forms a homo-oligomer. The three-dimensional structure of AbSGR-h was predicted by a deep learning-based method, which was evaluated by protein structure quality evaluation programs while structural stability of wild-type and mutant forms were investigated through molecular dynamics simulations. Furthermore, in concordance with the results of enzyme assay, molecular docking concluded the significance of D34 in ligand interaction. By combining biochemical analysis and computational prediction, this study unveils the detailed structural characteristics of the enzyme, including the probable pocket of interaction and the residues of structural and functional importance. It also serves as a basis for further studies on Mg-dechelatase such as elucidation of its reaction mechanism or inhibitor screening.
Highlights
Chlorophyll is a pigment that plays a crucial role in absorption, transmission and transformation of light energy during photosynthesis
We observed the presence of a motif similar to an incomplete metal-ion-dependent adhesion site (MIDAS) motif at 31–36 (T-H-S-D-S-T) for the SGR homolog from Anaerolineae bacterium SM23_63 shown in boldface in the multiple sequence alignment
Despite technological advancement and sincere attempts, there remains a huge gap between the number of known sequences and experimentally derived structures available in the Protein Data Bank (PDB), highlighting the difficulties of structure elucidation by experimental methods like X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy [54]
Summary
Chlorophyll is a pigment that plays a crucial role in absorption, transmission and transformation of light energy during photosynthesis. The first step of the degradation process is the extraction of magnesium (Mg) ion from chlorophyll a to form pheophytin a by Mg-dechelatase encoded by the Stay-Green (SGR) gene, which is responsible for Mendel’s greencotyledon peas [4]. This reaction is strictly regulated to prevent the formation of detrimental photoreactive chlorophyll intermediates, serving as the rate-limiting step of the chlorophyll breakdown pathway [5,6].
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