Heterologous expression of the Rhodobacter capsulatus BchI, -D, and -H genes that encode magnesium chelatase subunits and characterization of the reconstituted enzyme

Robert D. Willows, Samuel I. Beale*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    59 Citations (Scopus)

    Abstract

    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 tool of BchD occurred at a stoichiometry of 4 BchI:l BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH.

    Original languageEnglish
    Pages (from-to)34206-34213
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume273
    Issue number51
    DOIs
    Publication statusPublished - 18 Dec 1998

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