Rieske oxygenases perform precise C–H bond functionalization reactions in anabolic and catabolic pathways. These reactions are typically characterized as monooxygenation or dioxygenation reactions, but other divergent reactions are also catalyzed by Rieske oxygenases. Chlorophyll(ide) a oxygenase (CAO), for example is proposed to catalyze two monooxygenation reactions to transform a methyl-group into the formyl-group of Chlorophyll b. This formyl group, like the formyl groups found in other chlorophyll pigments, tunes the absorption spectra of chlorophyllb and supports the ability of several photosynthetic organisms to adapt to environmental light. Despite the importance of this reaction, CAO has never been studied in vitro with purified protein, leaving many open questions regarding whether CAO can facilitate both oxygenation reactions using just the Rieske oxygenase machinery. In this study, we demonstrated that four CAO homologues in partnership with a non-native reductase convert a Chlorophyll a precursor, chlorophyllidea, into chlorophyllideb in vitro. Analysis of this reaction confirmed the existence of the proposed intermediate, highlighted the stereospecificity of the reaction, and revealed the potential of CAO as a tool for synthesizing custom-tuned natural and unnatural chlorophyll pigments. This work thus adds to our fundamental understanding of chlorophyll biosynthesis and Rieske oxygenase chemistry.

中文翻译：

---

Rieske 加氧酶催化叶绿素 b 生物合成中的 C-H 键功能化反应

Rieske 加氧酶在合成代谢和分解代谢途径中进行精确的 C-H 键功能化反应。这些反应通常被表征为单加氧或双加氧反应，但其他不同的反应也由 Rieske 加氧酶催化。例如，叶绿素 (ide) a加氧酶 (CAO) 被提议用于催化两个单加氧反应以将甲基基团转化为叶绿素b的甲酰基基团。与其他叶绿素色素中的甲酰基一样，这种甲酰基可以调节叶绿素b的吸收光谱并支持几种光合生物适应环境光的能力。尽管这种反应很重要，但从未用纯化的蛋白质在体外研究过 CAO，这留下了许多悬而未决的问题，即 CAO 是否可以仅使用 Rieske 加氧酶机制促进这两种氧化反应。在这项研究中，我们证明了四种 CAO 同源物与非天然还原酶合作将叶绿素a前体叶绿素a转化为叶绿素b体外。对该反应的分析证实了所提出的中间体的存在，突出了反应的立体特异性，并揭示了 CAO 作为合成定制调整的天然和非天然叶绿素色素的工具的潜力。因此，这项工作增加了我们对叶绿素生物合成和 Rieske 加氧酶化学的基本理解。