Insights into the structure and function of the rate-limiting enzyme of chlorophyll degradation through analysis of a bacterial Mg-dechelatase homolog

Dey, Debayan; Dhar, Dipanjana; Fortunato, Helena; Obata, Daichi; Tanaka, Ayumi; Tanaka, Ryouichi; Basu, Soumalee; Itô, Hisashi

doi:10.1016/j.csbj.2021.09.023

Cited by 4 publications

(24 citation statements)

References 54 publications

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“…Thus, H32 and D34 residing in this region are more flexible than those in the computationally predicted structure. In fact, this result is in agreement with the previous study where root mean square fluctuation for individual residues obtained by molecular dynamics simulation showed that the site of D34 is flexible 27 . Although H32 and D34 positions were slightly different, the Cα‐atoms of D62 were found in the same position for both structures.…”

Section: Discussionsupporting

confidence: 93%

“…The crystal and computationally predicted structure were found to be almost identical. In our previous study, mutation in R26, Y28, T29, and D114 rendered the recombinant protein insoluble, suggesting that these residues are essential for maintaining the AbSGR‐h structure form 27 . The positions of these residues were correctly predicted in the computationally derived structure.…”

Section: Discussionmentioning

confidence: 80%

“…According to a previous study, AbSGR-h was assumed to exist as a hexameric complex in solution. 27 Major peaks were detected in the elution of gel filtration at the same position for the WT and three mutant AbSGR-h (D34E, D34N, and D34Q), indicating the presence of the hexameric form of proteins. This observation further confirms the role of D34 to be mainly catalytic rather than maintaining the multimeric conformation of the protein.…”

Section: Protein Expression and Purificationmentioning

confidence: 89%

“…In our previous study, mutation in R26, Y28, T29, and D114 rendered the recombinant protein insoluble, suggesting that these residues are essential for maintaining the AbSGR-h structure form. 27 The positions of these residues were correctly predicted in the computationally derived structure. However, the arrangements of the active site were slightly different (Figure 7).…”

Section: Substrate Docking Analysismentioning

confidence: 92%

“…Our previous study showed that three residues-H32, D34, and D62 in the AbSGR-h protein are critical for its catalytic activity, in which D34 is suggested to be involved in direct interaction with Chl a. 27 Additionally, the presence of D34 on the surface of protein surrounded by a hydrophobic patch of residues, provides an ideal environment for interaction with the substrate. Since the main objective of this study is to delve deep into the reaction mechanism of Mg-dechelatase enzyme, three mutations on the aforementioned D34 residue were prepared to understand the effect of substitutions on the catalytic ability of AbSGR-h. D34 was changed to glutamate (D34E) to check whether replacement with a similar property amino acid altered the activity of AbSGR-h.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

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Crystal structure and reaction mechanism of a bacterial Mg‐dechelatase homolog from the Chloroflexi Anaerolineae

et al. 2022

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Chlorophyll degradation plays a myriad of physiological roles in photosynthetic organisms, including acclimation to light environment and nutrient remobilization during senescence. Mg extraction from chlorophyll a is the first and committed step of the chlorophyll degradation pathway. This reaction is catalyzed by the Mg‐dechelatase enzyme encoded by Stay‐Green (SGR). The reaction mechanism of SGR protein remains elusive since metal ion extraction from organic molecules is not a common enzymatic reaction. Additionally, experimentally derived structural information about SGR or its homologs has not yet been reported. In this study, the crystal structure of the SGR homolog from Anaerolineae bacterium was determined using the molecular replacement method at 1.85 Å resolution. Our previous study showed that three residues—H32, D34, and D62 are essential for the catalytic activity of the enzyme. Biochemical analysis involving mutants of D34 residue further strengthened its importance in the functioning of the dechelatase. Docking simulation also revealed the interaction between the D34 side chain and central Mg ion of chlorophyll a. Structural analysis showed the arrangement of D34/H32/D62 in the form of a catalytic triad that is generally found in hydrolases. The probable reaction mechanism suggests that deprotonated D34 side chain coordinates and destabilizes Mg, resulting in Mg extraction. Besides, H32 possibly acts as a general base catalyst and D62 facilitates H32 to be a better proton acceptor. Taken together, the reaction mechanism of SGR partially mirrors the one observed in hydrolases.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 80%

Section: Protein Expression and Purificationmentioning

confidence: 89%

Section: Substrate Docking Analysismentioning

confidence: 92%

Section: Protein Expression and Purificationmentioning

confidence: 99%

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Crystal structure and reaction mechanism of a bacterial Mg‐dechelatase homolog from the Chloroflexi Anaerolineae

et al. 2022

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In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae

Sato,

Hirose,

Tanaka

et al. 2024

Photosynth Res

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In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

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Activity examination of plant Mg-dechelatase and its bacterial homolog in plants and in vitro

Ando,

Tanaka,

Ito

2024

Plant Physiology and Biochemistry

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Insights into the structure and function of the rate-limiting enzyme of chlorophyll degradation through analysis of a bacterial Mg-dechelatase homolog

Abstract: Graphical abstract

Cited by 4 publications

References 54 publications

Crystal structure and reaction mechanism of a bacterial Mg‐dechelatase homolog from the Chloroflexi Anaerolineae

Crystal structure and reaction mechanism of a bacterial Mg‐dechelatase homolog from the Chloroflexi Anaerolineae

In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae

Activity examination of plant Mg-dechelatase and its bacterial homolog in plants and in vitro

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