Kinetic And Catalytic Analysis Of The Magnesium Chelatase In Plants

The metabolic pathway known as (bacterio) chlorophyll biosynthesis is initiated by magnesium chelatase. This first step involves insertion of magnesium into protoporphyrinIX (proto), a process requiring ATP hydrolysis. Magnesium chelatase was composed of three subunits called BchI/ChlI, BchD/ChlD, and BchH/ChlH (40-45,70-90, and140-150kDa, respectively). Magnesium chelatase is not only catalysis the synthesis of chlorophyll, not also involve in plastid-to-nucleus retrograde signaling and ABA signaling, even regulate the gene expression. Despite recent progress from Rhodobacter capsulatus magnesium chelatase, however the characterization of Mg-chelatase in higher plant remains to be elucidated. Therefore, studying the kinetic analysis and then mechanism of magnesium chelatase in plant are very important to know the regulation of chlorophyll synthesis pathway, plastid-to-nucleus retrograde signaling and ABA signaling in chloroplast.In this study, we focused on magnesium chelatase in rice, set a new model system to investigate the function of magnesium chelatase in vivro, system analyzed the kinetic properites of all subunit and substrates and gained the reaction mechanism of magnesium chelatase. The main results in this study are as following:Cloned, expressed and purified all the subunit and cofactor protein GUN4of magnesium chelatase, set a new model system to investigate the function of magnesium chelatase in vivro. GUN4stimulate magnesium chelatse by associate H subunit to form a stable complex with Proto. The stimulatory effect of GUN4was magnesium and ATP-depenged and increased the reaction rate16-fold. The C-terminal9or10amino acids, conserved in eukaryotic GUN4proteins but absent from cyanobacterial GUN4proteins, are essential for activation of the magnesium chelatase, but not for porphyrin binding. D subunit form a hexamer by itself, while I subunit as monomer or dimer in vivo. GUN4and H subunit form a complex in vivo, which molecular mass are180kDa, using BN/SDS-PAGE, gel filtration and western blotting.Michaelis-menten kinetics was observed with the substrate ATP and Proto, whereas the magnesium exhibited sigmoidal kinetics. A sigmoidal relationship was observed with H subunit as the substrate, indicating cooperativity of H subunit. The data of GUN4and I subunit fitted to the Michaelis-Menten equation. A Hill coefficient of3suggests that more than three H subunit and GUN4complex bind per ID complex. Formation of the GUN4-ChlH-Proto complex is a rate-limiting step in magnesium chelatase.CHLI2can not substitute for CHLI1. ChlI2is inactive and poisons the activity of ChlI1.