The CHLI1 Subunit of Arabidopsis thaliana Magnesium Chelatase Is a Target Protein of the Chloroplast Thioredoxin

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.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...

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis

“…Recombinant TRX m and TRX f reduced intramolecular disulfide bond formation at the C terminus of the MgCh subunit CHLI and stimulated the ATPase activity of CHLI (Ikegami et al, 2007;Luo et al, 2012) as well as MgCh activity in vitro (Luo et al, 2012). The physical interaction of TRX f with CHLI was confirmed by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays (Luo et al, 2012).…”

“…Numerous in vitro and in vivo studies have shown that TRX f and TRX m are required for multiple metabolic processes in chloroplasts, including the Calvin-Benson cycle (Collin et al, 2003;Michelet et al, 2013;Okegawa and Motohashi, 2015;Yoshida et al, 2015), ATP synthesis (Schwarz et al, 1997) and NADPH export (Wolosiuk et al, 1979;Lara et al, 1980), starch metabolism (Fu et al, 1998;Mikkelsen et al, 2005;Seung et al, 2013;Thormählen et al, 2013), fatty acid synthesis (Sasaki et al, 1997), amino acid synthesis (Balmer et al, 2003), and chlorophyll (Chl) synthesis (Ikegami et al, 2007;Luo et al, 2012). Functional genetic analyses have shown that TRX m4 knockout mutants were affected in cyclic electron flow around PSI (Courteille et al, 2013), while a TRX m3 knockout mutant was affected in meristem development (BenitezAlfonso et al, 2009).…”

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

“…However, given the Trx ability to reduce CHLI in vitro (Fig. 2 E and F) (22) and the physical interaction between Trx-f and CHLI in vivo (33), the FTR/Trx pathway is thought to contribute to CHLI redox regulation possibly in a redundant manner. It is highly plausible that the severely chlorotic phenotype of ftrb ntrc is a consequence of disrupted redox regulation of chlorophyll biosynthesis via both the FTR/Trx and NTRC pathways.…”

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms

“…One of the most obvious parameters differing in plastid extracts incubated in light and dark is the redox status. Because the well-known STN7 kinase was shown to be regulated by a redox shift within the photosynthetic machinery (Bellafiore et al, 2005;Bonardi et al, 2005;Rochaix et al, 2012) and enzymatic activities of TBS are regulated in a redox-dependent manner (Jensen et al, 2000;Ikegami et al, 2007;Luo et al, 2012;Pérez-Ruiz et al, 2014), it was assumed that the GUK and thus GUN4 phosphorylation could be regulated in a Figure 8. Significance of the extended C-terminal segment of GUN4 and conservation of the phosphorylation site in angiosperms.…”