Heterologous Expression of the Barley (Hordeum vulgareL.)Xantha-f, -gand -hGenes that Encode Magnesium Chelatase Subunits

Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg(2+)into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are namedXantha-h,Xantha-gandXantha-fin barley (Hordeum vulgareL.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins ofRhodobacter capsulatus,Synechocystissp. PCC6803 andThermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genesXantha-h,-gand -fwere cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins inEscherichia coliBL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.