Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits

Abstract

AbstractBiosynthesis 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 Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). 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 of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus 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 genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(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.