Dey Debayan, Tanaka Ryouichi, Ito Hisashi
Graduate School of Life Science, Hokkaido University, N10 W8, Sapporo, 060-0810, Japan.
Institute of Low Temperature Science, Hokkaido University, N19 W8, Sapporo, 060-0819, Japan.
J Mol Evol. 2023 Apr;91(2):225-235. doi: 10.1007/s00239-023-10100-9. Epub 2023 Mar 3.
Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.
叶绿素酸酯a加氧酶（CAO）负责在两步氧化反应中将叶绿素a转化为叶绿素b。CAO属于 Rieske-单核铁加氧酶家族。尽管已描述了其他 Rieske 单加氧酶的结构和反应机制，但植物 Rieske 非血红素铁依赖性单加氧酶家族的一个成员尚未进行结构表征。该家族中的酶通常形成三聚体结构，电子在非血红素铁位点和相邻亚基的 Rieske 中心之间转移。CAO 应该形成类似的结构排列。然而，在诸如微拟球藻和聚球藻等绿藻纲中，CAO 由两个基因编码，其中非血红素铁位点和 Rieske 簇位于不同的多肽上。尚不清楚它们是否能形成类似的结构组织以实现酶活性。在本研究中，通过基于深度学习的方法预测了模式植物拟南芥和绿藻微拟球藻中CAO的三级结构，随后对预测模型进行能量最小化和后续的立体化学质量评估。此外，预测了微拟球藻CAO表面的叶绿素a结合腔以及作为电子供体的铁氧还蛋白的相互作用。预测了微拟球藻CAO中的电子转移途径，尽管它形成异二聚体复合物，但CAO活性位点的整体结构是保守的。本研究中呈现的结构将为理解CAO所属的植物单加氧酶家族的反应机制和调控提供基础。
