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.
Willows
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Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Beale
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D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005798:Genes, Bacterial; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011524:Protoporphyrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics*; D013053:Spectrophotometry
D017931:DNA Primers; D046911:Macromolecular Substances; D011524:Protoporphyrins; D011994:Recombinant Proteins; C028025:protoporphyrin IX; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium