ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artifact
Résumé
Magnesium chelatase inserts Mg 2+} into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of ATPases Associated with various cellular Activities (AAA +}), but only I hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In this work we demonstrate that gel filtration chromatography of affinity purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high and a low molecular weight fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high molecular weight fraction and magnesium chelatase activity was only associated with the low molecular fraction. We demonstrated that light converted monomeric low molecular weight H-subunit into high molecular weight aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.
Origine : Fichiers produits par l'(les) auteur(s)