Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein

Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes , three genes - BchI, D and H - encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characteriz ed. Since the N-terminal half of the D subunit is homologous to the I subun it, the C-terminal portion of the pea D was used for antigen production. Th e antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelata se activity. Expression in Escherichia coli with a construct for the full-l ength protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubiliz ed in 6 M urea. However, when host cells were co-transformed with expressio n vectors for the full-length D subunit and for the 70 kDa HSP chaperonin p rotein, a substantial portion of the 89 kDa protein was expressed in a solu ble form which was active in a Mg-chelatase reconstitution assay.