C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis

Oryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg2+ and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension.Structured summary of protein interactionsChlH and ChlHbind by molecular sieving (View interaction)ChlH and ChlHbind by molecular sieving (View interaction)ChlD and ChlDbind by molecular sieving (View interaction)

► Oryza sativa GUN4, ChlI, ChlD, and ChlH reconstituted Mg-chelatase activity. ► Preactivation of ChlH with GUN4, protoporphyrin and Mg2+ gave maximum activity. ► Protoporphyrin omission during preactivation almost completely abolished activity. ► Protoporphyrin preincubation with OsChlH was most important for preactivation. ► GUN4’s C-terminal is essential for preactivation of ChlH but not porphyrin binding.