Magnesium chelatase from Rhodobacter sphaeroides: initial characterizationof the enzyme using purified subunits and evidence for a BchI-BchD complex

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses t he insertion of Mg into protoporphyrin IX, the first committed step in (bac terio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His(6) affin ity tags, which has led to the production of large amounts of highly purifi ed, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2, and ATP, yielding highly active protein. BchI/B chD mixtures prepared in this way were used in conjunction with BchH to det ermine the kinetic parameters of R. sphaeroides Mg chelatase for its natura l substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish a nd maintain this complex. Gel filtration data suggest that BchI and BchD fo rm a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained i n its correct conformation in the presence of BchI, Mg2+ and ATP.