MG-CHELATASE OF TOBACCO - IDENTIFICATION OF A CHL-D CDNA SEQUENCE ENCODING A 3RD SUBUNIT, ANALYSIS OF THE INTERACTION OF THE 3 SUBUNITS WITH THE YEAST 2-HYBRID SYSTEM, AND RECONSTITUTION OF THE ENZYME-ACTIVITYBY COEXPRESSION OF RECOMBINANT CHL-D, CHL-H AND CHL-I

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium io n into protoporphyrin IX, the last common intermediate precursor in ch lorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhod obacter 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 prote ins are necessary for the Mg chelation, and genes homologous to bchH a nd bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characteriz ed. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three s ubunits of one plant species. The CHL D polypeptide deduced from the o pen 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 pepti de sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are li nked with a glutamine/asparagine/proline-rich region flanked by a high ly acid-rich segment. Protein-protein interaction among the three subu nits CHL D, H and was studied using the yeast two-hybrid system. Physi cal interaction was demonstrated between CHL D and CHL I indicating th at CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H w ith CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicat ed in the more sensitive assay on X-Gal-containing agar plates. In vit ro Mg2+ insertion into protoporphyrin IX was demonstrated in protein e xtracts of yeast strains expressing the three subunits of tobacco Mg-c helatase. The reconstitution of the recombinant enzyme activity requir ed additional ATP.