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Mg‐chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two‐hybrid system, and reconstitution of the enzyme activity by co‐expression of recombinant CHL D, CHL H and CHL I
Author(s) -
Papenbrock Jutta,
Gräfe Susanna,
Kruse Elisabeth,
Hänel Frank,
Grimm Bernhard
Publication year - 1997
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1997.12050981.x
Subject(s) - biochemistry , biology , peptide sequence , protein subunit , complementary dna , open reading frame , transmembrane domain , amino acid , microbiology and biotechnology , gene
Summary Mg‐protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg‐protoporphyrin IX. In Rhodobacter sphaeroides , and Synechocystis , the three open reading frames bchD/chlD , bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg‐chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full‐length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline‐rich region flanked by a highly acid‐rich segment. Protein‐protein interaction among the three subunits CHL D, H and I was studied using the yeast two‐hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg‐chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X‐Gal‐containing agar plates. In vitro Mg 2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg‐chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.