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.

Figure 1

Formyl groups are abundant modifications made on the chlorophyll (Chl) scaffold. (a) The pigments Chl b, d, and f each bear formyl groups on their macrocyclic chlorin scaffolds at various positions. These modifications contribute to their characteristic absorbance patterns. The modifications found in Chl d and f are formed using an as-yet unidentified enzyme and the photooxidoreductase Chl f synthase,⁻ respectively. Formation of the C7 formyl group in chlorophyll(ide) b is instead proposed to be installed via two sequential chlorophyllide a oxygenase (CAO)-catalyzed reactions that transform the C7-methyl group of chlorophyll(ide) a into the formyl group of chlorophyll(ide) b through C7-hydroxymethyl and C7-dihydroxymethyl intermediates. All three chiral centers in Chl a, b, and intermediates are labeled with a red asterisk and for the chemical structure, R = H or C₂₀H₃₉ for chlorophyllide and chlorophyll pigments, respectively. This proposal for CAO is unlike that for other transformations that proceed through more than one monooxygenation reaction and require two Rieske oxygenases to be completed.⁻ This proposal is also unlike that needed to convert a methyl group into a formyl group in the catabolism of 4-toluene sulfonate, which requires both a Rieske oxygenase and dehydrogenase. (b) CAO has potential to be used as a tool for formylating the Chl scaffold to produce custom-tuned native and non-native pigments. (c) The CAO homologues studied in this work have different domain architectures. All homologues of CAO are predicted to have a Rieske [2Fe-2S] cluster and a mononuclear nonheme iron site in their catalytic domains (blue). These metallocenters are coordinated by two His and two Cys ligands and a facial triad of residues, respectively. The Micromonas pusilla CAO homologue is found in two polypeptide chains and the Arabidopsis thaliana and Chlamydomonas reinhardtii homologues have N-terminal regulatory domains (peach). As PhCAO appears to contain the simplest architecture of all four homologues, it was the first CAO homologue characterized in this work.

Figure 2

P. hollandica CAO can be purified and used for in vitro biochemical studies. A gel filtration profile of PhCAO reveals that CAO purifies with the expected trimeric quaternary structure of a Rieske oxygenase. An inset of an SDS-PAGE gel reveals the purity of the isolated trimeric PhCAO (expected monomeric molecular weight of 41 kDa).

Figure 3

CAO homologues transform Chlide a, not Chl a, into Chlide b in cell lysate. (a) Combination of a Chl pigment with recombinantly expressed and purified T. aestivum chlorophyllase (Chlase) permits formation of the desired Chlide a and Chlide b pigments. (b) Activity of chlorophyllase on Chl a and Chl b. Extracted ion chromatograms of the chlorophyllase activity assays product with Chl a (top trace) and Chl b (bottom trace). The LC-MS method designed and employed for pigment separation in this work relies on an acidic running solvent and causes loss of the central Mg²⁺ ion and the addition of two protons to the pigments under study. Therefore, the m/z = 593.2758 and m/z = 607.2551 represents the [M + H]⁺ of Chlide a and Chlide b minus Mg²⁺ plus 2H⁺, respectively (top panel). Chlorophyllase converts nearly 100% and 85% of Chl a and Chl b into their Chlide counterparts, respectively (bottom panel). (c) Proposed reaction scheme catalyzed by CAO in cell lysate. (d) None of the CAO homologues can transform Chl a into Chl b in A. thaliana cell lysate. (e) The extracted ion chromatograms for the CAO homologue reaction products when combined with a Chlide a substrate and A. thaliana cell lysate reveal that all four CAO homologues can convert Chlide a into Chlide b. Of note, the cell lysate, pyridine, and acid can each shift the diastereomer equilibrium of the Chlide b standard (traces 3–4 and 7). The black asterisk indicates the major peak of the standard, which is also observed in the assays and the red asterisk corresponds to the diastereomer peak observed in the enzymatic assays. (f) PhCAO shows the highest percent conversion among all the four homologues in the presence of A. thaliana cell lysate and a Chlide substrate. All data shown in the bar graphs was performed in duplicate and data are presented as mean values.

Figure 4

CAO homologues transform chlorophyllide a into chlorophyllide b in the presence of the non-native reductase VanB. (a) All four CAO homologues show the ability to convert Chlide a into Chlide b in E. coli cell lysate, suggesting that a non-native reductase may work with CAO to facilitate the reaction. PhCAO shows the highest percent conversion among all four homologues. (b) Reaction scheme of the CAO/VanB catalyzed conversion of Chlide a into Chlide b (left panel). Treatment of the Chlide b standard with acid or pyridine shows new diastereomer peaks. Similarly, the reaction product from the CAO homologue reactions can be converted into the main diastereomer of the Chlide b standard under acidic conditions (right panel). (c) The extracted ion chromatograms for the product formed when the CAO homologues are combined with Chlide a, VanB, and NADPH. This data shows a diastereomer of Chlide b is formed in these reactions. Again, PhCAO shows the highest percent conversion among all four homologues. (d) Reaction scheme for converting Chlide a into Chlide a′ with pyridine. (e) An extracted ion chromatogram of the Chlide a peak suggests the Chlide a′ sample shows a different diastereomer distribution than Chlide a. (f) PhCAO shows a higher percent conversion on Chlide a′ than Chlide a. In panels c and f, reactions were performed in triplicate, and in panels a and e, they were performed in duplicate. In all bar graphs presented, data are shown as mean values.

Figure 5

7-OH-Chlide a can be transformed into Chlide b by combination of the CAO homologues with VanB and NADPH. (a) Reaction scheme for synthetic conversion of Chl b into 7-OH-Chlide a. (b) Extracted ion chromatograms of chlorophyllase catalyzed hydrolysis of 7-OH-Chl a. The m/z = 609.2708 represents the [M + H]⁺ of 7-OH-Chlide a minus Mg²⁺ plus 2H⁺ (see Figure 3a). (c) Absorbance spectra of the Chlide a, Chlide b, and 7-OH Chlide a that were enzymatically and synthetically produced in this work. (d) Reaction scheme of the oxygenation reactions that are catalyzed by CAO. (e) Extracted ion chromatograms for the PhCAO reaction products reveals the formation of a 7-OH-Chlide a intermediate. Acid treatment refers to a screening that was performed over a range of different pH values. This screening revealed that when the pH was adjusted to a value of 2, all Chlide b appeared as one peak. (f) All four CAO homologues show the ability to transform the intermediate (7-OH-Chlide a) into the final product (Chlide b). Again, PhCAO shows the highest percent conversion among all four homologues.

Figure 6

3-formyl-Chlide a (Chlide d) can be transformed into 3-formyl-Chlide b by combination of PhCAO with VanB and NADPH. (a) Reaction scheme to synthesize 3-formyl-Chlide a and the proposed route to C7-oxygenation by CAO. (b) The extracted ion chromatograms for the product formed when Chlide a or Chlide a with β-mercaptoethanol and heme was provided to PhCAO as a substrate. (c) The extracted ion chromatograms show that PhCAO can transform 3-formyl-Chlide a (Chlide d) into 3-formyl-Chlide b.