Magnesium Chelatase Subunit Research Articles

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.

Heterologous Expression of the Rhodobacter capsulatus BchI, -D, and -H Genes That Encode Magnesium Chelatase Subunits and Characterization of the Reconstituted Enzyme

Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, and bchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH2-terminally His6-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 M urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH.

Chromosomal locations of six barley genes encoding enzymes of chlorophyll and heme biosynthesis and the sequence of the ferrochelatase gene identify two regulatory genes

The chromosomal location of six barley genes was determined by Southern blot experiments using wheat strains which contained the short or long arm from one of the seven barley chromosomes. The genes analysed encode enzymes that are required for chlorophyll and heme synthesis. Glutamyl-tRNA Glu synthetase, glutamate 1-semialdehyde 2,1-aminotransferase and glutamyl-tRNA Glu reductase are common to both pathways and catalyse the initial reactions from glutamate to 5-aminolevulinic acid. Magnesium chelatase and ferrochelatase are at the branch between chlorophyll and heme synthesis and insert magnesium and ferrous, respectively, into protoporphyrin IX. The chromosomal locations of the barley structural genes for the following enzymes were: glutamyl-tRNA Glu synthetase — short arm of chromosome 6 (6H); glutamate 1-semialdehyde 2,1-aminotransferase — β arm of chromosome 1 (7H); ferrochelatase — long arm of chromosome 7 (5H); magnesium chelatase subunit Xantha-F — short arm of chromosome 2 (2H); magnesium chelatase subunit Xantha-H — α arm of chromosome 1 (7H). Two glutamyl-tRNA Glu reductase genes were found in the barley genome and one was localised on the short arm of chromosome 5 (1H). It is suggested that the other reductase gene is located on the long arm of chromosome 5 (1H). The barley ferrochelatase gene consists of nine exons and was isolated and sequenced from wild type and two mutant barley, tigrina- d 12 and tigrina- o 34, and is located on chromosome 7 (5H). In all three cases, an identical sequence was obtained, excluding the possibility that the deregulated synthesis of 5-aminolevulinic acid in the two tigrina mutants results from mutations in the ferrochelatase gene.

ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides.

Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

Expression of the chlI, chlD, and chlH Genes from the Cyanobacterium Synechocystis PCC6803 in Escherichia coli and Demonstration That the Three Cognate Proteins Are Required for Magnesium-protoporphyrin Chelatase Activity

Magnesium-protoporphyrin chelatase catalyzes the first step unique to chlorophyll synthesis: the insertion of Mg2+ into protoporphyrin IX. Genes from Synechocystis sp. PCC6803 with homology to the bchI and bchD genes of Rhodobacter sp. were cloned using degenerate oligonucleotides. The function of these genes, putatively encoding subunits of magnesium chelatase, was established by overexpression in Escherichia coli, including the overexpression of Synechocystis chlH, previously cloned as a homolog of the Rhodobacter bchH gene. The combined cell-free extracts were able to catalyze the insertion of Mg2+ into protoporphyrin IX in an ATP-dependent manner and only when the products of all three genes were present. The ChlH, ChlI, and ChlD gene products are therefore assigned to the magnesium chelatase step in chlorophyll a biosynthesis in Synechocystis PCC6803. The primary structure of the Synechocystis ChlD protein reveals some interesting features; the N-terminal half of the protein shows 40-41% identity to Rhodobacter BchI and Synechocystis ChlI, whereas the C-terminal half displays 33% identity to Rhodobacter BchD. This suggests a functional as well as an evolutionary relationship between the "I" and "D" genes.

Cloning, Subcellular Localization and Expression of chl1, a Subunit of Magnesium Chelatase in Soybean

Mg-insertion is the first committed step in chlorophyll synthesis and is catalyzed by Mg-chelatase. In photosynthetic bacteria, bchI gene product was suggested to be a subunit of Mg-chelatase. We isolated a bchI homolog from a soybean cDNA library and designated it as chlI. CHLI consisted of 421 amino acid residues and the sequence exhibited a high similarity to other BchI homologs. CHLI contained an ATP-binding motif found in other BchI homologs. CHLI was localized in the soluble fraction in soybean chloroplasts, suggesting that it was stromal subunit of Mg-chelatase. chlI mRNA in cell culture (SB-P) of soybean was reversibly induced by light.