Filament Formation by ChlI Challenges the Current View of Magnesium Chelatase Architecture

Magnesium chelatase (MgCh) catalyzes the first committed step in chlorophyll biosynthesis by inserting Mg2+ into the tetrapyrrole ring. The enzyme comprises three core subunits: ChlI, ChlD, and ChlH, and requires the auxiliary factor GUN4 for full activity. Despite extensive investigation, the structural organization of the active holoenzyme and the mechanistic coupling between ATP hydrolysis and Mg2+ insertion remain poorly defined. Here, we used cryo-electron microscopy to investigate MgCh architecture. We show that both cyanobacterial and plant ChlI homologs assemble into filamentous helical structures in the presence of Mg2+ and either ATP or ADP. However, only ATP-induced oligomers are susceptible to disassembly by ChlD. Structural analysis of the ATP-driven assemblies reveals compact inter-subunit packing, with ADP and Mg2+ coordinated at the interfacial regions. These findings suggest that ATP hydrolysis promotes subunit compaction and may facilitate partial dehydration of the Mg2+ hydration shell. Low-resolution reconstructions further provide a structural framework for ChlD engagement with ChlI filaments. Finally, we demonstrate that phosphatidylglycerol enhances catalytic activity, supporting a role for membrane lipids in modulating MgCh function. Significance statementMagnesium chelatase catalyzes the first committed step of chlorophyll biosynthesis, yet the structural basis of its activation and coupling to ATP hydrolysis remains unclear. Using cryo-electron microscopy, we show that the ATPase subunit ChlI forms filamentous helical oligomers in the presence of Mg2+ and nucleotide, a property conserved across cyanobacterial and plant homologs but not previously recognized. Only oligomers generated through ATP hydrolysis interact efficiently with the ChlD subunit, indicating that hydrolysis produces a distinct, recognition-competent conformation. Structural analysis further suggests that ATP hydrolysis promotes subunit compaction and partial dehydration of Mg2+. Together, these findings reveal a previously unrecognized oligomeric state of ChlI and provide a structural framework for understanding how ATP hydrolysis regulates magnesium chelatase activity.