RECONSTITUTION OF AN ACTIVE MAGNESIUM CHELATASE ENZYME COMPLEX FROM THE BCHI, BCHD, AND BCHH GENE-PRODUCTS OF THE GREEN SULFUR BACTERIUM CHLOROBIUM-VIBRIOFORME EXPRESSED IN ESCHERICHIA-COLI

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (ba cterio)chlorophyll-specific branch of the porphyrin biosynthetic pathw ay, catalyzes the insertion of Mg2+ into protoporphyrin IX, Three gene s, designated bchI, -D, and -H, from the strictly anaerobic and obliga tely phototrophic green sulfur bacterium Chlorobium vibrioforme show a significant level of homology to the magnesium chelatase-encoding gen es bchI, -D, and -H and chlI, -D, and -H of Rhodobacter sphaeroides an d Synechocystis strain PCC6803, respectively, These three genes were e xpressed in Escherichia coli; the subsequent purification of overprodu ced BchI and -H proteins on an Ni2+-agarose affinity column and denatu ration of insoluble BchD protein in 6 RI urea were required for recons titution of Mg-chelatase activity in vitro, This work therefore establ ishes that the magnesium chelatase of C. vibrioforme is similar to the magnesium chelatases of the distantly related bacteria R. sphaeroides and Synechocystis strain PCC6803 with respect to number of subunits a nd ATP requirement, In addition, reconstitution of an active heterolog ous magnesium chelatase enzyme complex was obtained by combining the C , vibrioforme BchI and -D proteins and the Synechocystis strain PCC680 3 ChlH protein, Furthermore, two versions, with respect to the N-termi nal start of the bchI gene product, were expressed in E. coli, yieldin g ca, 38- and ca, 42-kDa versions of the BchI protein, both of which p roved to be active, Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expres sed proteins, are also present in C, vibrioforme.