Abstract
Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.
Highlights
Chlorophylls are the most abundant light-harvesting pigments on Earth and essential for the process of photosynthesis
Chlorophyll biosynthesis is a major anabolic pathway divided into 15 enzymatic steps, each catalyzed by a unique enzyme [1]
We report a completely recombinant assay based on the barley Xantha-l gene product encoding the aerobic cyclase XanL
Summary
Chlorophylls are the most abundant light-harvesting pigments on Earth and essential for the process of photosynthesis. They belong to a large family of tetrapyrrole molecules including hemes, bilins, and corrins. Chlorophylls are distinguished from the other tetrapyrroles by a centrally chelated magnesium ion and a fifth isocyclic ring, named the E ring. Chlorophyll biosynthesis is a major anabolic pathway divided into 15 enzymatic steps, each catalyzed by a unique enzyme [1]. The initial nine biosynthetic reactions are common to both chlorophyll and heme. In the first unique step of the chlorophyll biosynthetic pathway, Mg2+ is inserted into protoporphyrin IX. A methyl group is transferred to the carboxyl group of the propionate on the C ring of Mg-protoporphyrin IX, generating Mg-protoporphyrin IX monomethyl ester (MPE), which is the substrate of the MPE cyclase
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