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.
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/chID, bchH/chIH and bchI/chII 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 bchI 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 Mg2+ 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.
Papenbrock
J
J
Institut für Pflanzengenetik und Kulturpflanzenforschung, Abt. Für Molekulare Zellbiologie, Gatersleben, Germany.
Gräfe
S
S
Kruse
E
E
Hänel
F
F
Grimm
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics; D008190:Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008190:Lyases; C086788:magnesium chelatase; C100607:ChlD protein, Synechocystis sp. PCC 6803