Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

^*Hans-Knöll-Institut für Naturstoff-Forschung e.V., Department of Cell and Molecular Biology, Beutenbergstr. 11, 07745 Jena, Germany; and ^†Institut für Pflanzengenetik und Kulturpflanzenforschung, AG Chlorophyll Biosynthesis, Corrensstr. 3, 06466 Gatersleben, Germany

Edited by Lawrence Bogorad, Harvard University, Cambridge, MA, and approved December 21, 1998 (received for review July 20, 1998)

Abstract

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Footnotes

↵ ‡ To whom reprint requests should be addressed. e-mail: fhaenel{at}pmail.hki-jena.de.
This paper was submitted directly (Track II) to the Proceedings Office.
ABBREVIATIONS:

Chl,

chlorophyll;

Bch,

bacteriochlorophyll;

GST,

glutathione S-transferase;

N,

N terminal;

NM1,

N terminal and motif;

NM1LM2,

N terminal, motif, linker region, and motif;

M1LM2,

motif, linker region and motif;

M1LM2C,

motif, linker region, motif, and C terminal;

M2C,

motif and C terminal;

C,

C terminal;

AD,

activating domain;

BD,

binding domain