Abstract
Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370-375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.
Highlights
The reactions encompassing higher plant chlorophyll biosynthesis consist of 12 enzymes that work in unison to produce chlorophyll a from 5-aminolevulinic acid and constitute one of the most important pathways for chloroplast biogenesis
Macroarray analysis of the genes involved in tetrapyrrole biosynthesis indicates that the expression of CHLI1 is higher than CHLI2 under both light and dark growth conditions [11]
Magnesium chelatase activity is known to be sensitive to thiol group reagents (26 –29): Synechocystis magnesium chelatase activity is dependent on DTT concentrations, and the thiol modifying reagent NEM bound to CHLI inhibits its ATPase activity [30]
Summary
The reactions encompassing higher plant chlorophyll biosynthesis consist of 12 enzymes that work in unison to produce chlorophyll a from 5-aminolevulinic acid and constitute one of the most important pathways for chloroplast biogenesis. In (bacterio)chlorophyll a-producing prokaryotes [1], chlorophyll a-synthesizing bacteria, and higher plants, the magnesium chelatase complex consists of ϳ40-, ϳ70-, and ϳ140-kDa subunits named I, D, and H respectively [2, 3]. The stoichiometry of these subunits within the magnesium chelatase complex as well as its complete three-dimensional structure is yet to be determined. Thioredoxin Regulates Magnesium Chelatase Activity results in the production of chlorophyll or the insertion of Fe2ϩ produces heme, this enzyme is a primary candidate for the regulation of overall tetrapyrrole biosynthesis pathway. Mg2ϩ concentrations in chloroplasts are low, and magnesium chelatase is essentially inactive, whereas the Mg2ϩ concentrations increase in the light, and the enzymes become active, permitting the flux of the substrate, protoporphyrin IX, into the chlorophyll biosynthetic pathway
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