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.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. Among the chlorophyll biosynthetic enzymes, magnesium chelatase (EC 6.6.1.1) catalyzes the insertion of Mg 2ϩ into protoporphyrin IX, the first dedicated step in chlorophyll biosynthesis. 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. From the biochemical analysis using recombinant subunits of magnesium chelatase from the photosynthetic bacteria Rhodobacter and cyanobacterium Synechocystis sp. PCC6803, it was determined that the largest H subunit binds protoporphyrin IX noncovalently (4), suggesting that the H subunit catalyzes the metal chelation reaction. Although ATP and Mg 2ϩ are required for the interaction between I and D subunits (5, 6), I-D complex formation is independent from the ATP hydrolysis activity of I subunit. In contrast, the ATP hydrolysis process is required for...
The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.
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