Functional Studies Of Carboxyl-terminal Of Oryza Sativa GUN4,a Regulatory Protein Of Magnesium Chelatase

Higher plants produce four classes of tetrapyrroles, namely, chlorophyll (Chl), heme, siroheme, and phytochromobilin. Tetrapyrroles play essential roles in a wide range of biological activities. Chls are the most abundant tetrapyrroles, and essential for growth and development of green plants. The first enzyme in the chlorophyll biosynthetic pathway (Mg2+branch) is magnesium chelatase. It catalyzes insertion of magnesium into protoporphyrin IX (proto), and requires ATP hydrolysis. It consists of three subunits:I subunit (ChlI), D subunit (ChlD), and H subunit (ChlH). These subunits not only form a complex to accomplish the reaction but also are involved in other metabolic pathways. GUN4is a porphyrin-binding protein. It can activate the activity of magnesium chelatase. Our previous studies found that the C-terminal several amino acids of GUN4are important, this amino acid sequence is conserved in higher plants, and is absent in the cyanobacteria. so in higher plants, GUN4may have a special regulatory way.Our study is focused on the interaction of OsGUN4and OsChlH of magnesium chelatase in vitro, and activity analysis of magnesium chelatase, mainly includes two aspects:First, we removed the C-terminal8amino acid residues by deletion mutation and obtained the mutated OsGUN4L. Yeast-two-hybrid system and GST-Pull down assays showed that OsGUN4L can still interact with OsChlH. So OsGUN4lacking the C-terminal amino acids can still bind OsChlH.Secondly, we expressed and purified OsGUN4and OsGUN4L in vitro, and analyzed the activity of magnesium chelatase. We found that OsGUN4L lost the activation ability to magnesium chelatase.In summary, the C-terminal amino acids deletion of OsGUN4can still interact with OsChlH, but can’t activate the activity of magnesium chelatase. This study will help in further clarifying the mechanism of magnesium chelatase and deepening the understanding of multi-subunit enzymatic reaction.