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 characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The 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-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.
Luo
M
M
Department of Biological Sciences, Clemson University, SC 29634-1903, USA.
Weinstein
J D
JD
Walker
C J
CJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism; D005591:Chemical Fractionation; D002736:Chloroplasts / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D007118:Immunoassay; D008190:Lyases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D018833:Chaperonins; D008190:Lyases; C086788:magnesium chelatase