Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins Bchl, BchD, and BchH to catalyze the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that Bchl forms hexamers and belongs to the ATPases associated with various cellular activities (AAA(+)) family of proteins. AAA(+) proteins are Mg2+-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient Bchl subunits, we demonstrate that binding of ATP is sufficient to form Bchl oligomers. Further, ATPase-deficient Bchl proteins can form mixed oligomers with WT Bchl. The formation of Bchl oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT Bchl with ATPase-deficient Bchl in an assay disrupts the chelatase reaction, but the presence of deficient Bchl does not inhibit ATPase activity of the WT Bchl. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each Bchl within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient Bchl are unable to perform this ATIP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.