Rieske Oxygenase Catalyzed C–H Bond Functionalization Reactions in Chlorophyll b Biosynthesis

Abstract

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.

The in Vitro Biosynthesis of Chlorophyll b via Enzyme Catalysis

“…However, the efficient biosynthesis of high-value biomolecules in vitro is still underexplored. 1 As chlorophyll(ide) pigments are extensively used in the cosmetic, food, agricultural, and pharmaceutical industries, 2 the custom-tuned synthesis of natural and unnatural chlorophyll-(ide) derivatives in vitro is desirable. In this issue of ACS Central Science, Jennifer Bridwell-Rabb and co-workers employ four chlorophyll(ide) a oxygenase (CAO) homologues in cooperation with a non-native reductase to convert chlorophyllide a (Chlide a) into chlorophyllide b (Chlide b) in vitro.…”

“…In this issue of ACS Central Science, Jennifer Bridwell-Rabb and co-workers employ four chlorophyll(ide) a oxygenase (CAO) homologues in cooperation with a non-native reductase to convert chlorophyllide a (Chlide a) into chlorophyllide b (Chlide b) in vitro. 1 Chlorophylls are a class of naturally occurring pigments and serve key roles in photosynthetic organisms; 1,3 however, their synthesis in vitro is complicated due to a lack of efficient protocols for in vitro reconstitution of an enzyme and demonstration of its activity in isolation. 3 With the increasing demand for green and sustainable chemistry, chemists are pursuing those desirable systems that exclude noble metals, harsh conditions, toxic reagents, etc.…”

“…to prohibit electrons from reacting with the Chlide a substrate, the protein, or an activated oxygen intermediate. 1 The investigation of the substrate scope of Chlide a suggested the lack of a phytol chain, the presence of a central metal ion, and the electronics of the substrate may be important for achieving an efficient reaction with CAO.…”

“…(2) The MS experiments indicated 7-OH-Chlide a is a true intermediate of the oxygenation reaction (Figure 1c). 1 Regarding the reductase system, we anticipate that exogenous light or electricity could serve as a potentially green, Earth-abundant, and sustainable reductant to replace the reductase, which might improve the yields, 7 extend the substrate scopes, and generate new selectivity. Also, CAO has an obvious preference for the Chlide a′ diastereomer, 8 indicating that the stereochemistry in enzyme catalysis is also crucial.…”

“…Enzyme catalyzed chemical transformations provide sustainable strategies and enable synthetic approaches based on their powerful reactivity and potential for unique selectivity. However, the efficient biosynthesis of high-value biomolecules in vitro is still underexplored . As chlorophyll­(ide) pigments are extensively used in the cosmetic, food, agricultural, and pharmaceutical industries, the custom-tuned synthesis of natural and unnatural chlorophyll­(ide) derivatives in vitro is desirable.…”