Magnesium Chelatase - a Key Enzyme in Chlorophyll Biosynthesis

Abstract: The biosynthesis of chlorophylls is an important process in nature. The green pigments play a fundamental role in absorbing and transferring solar energy to the photosynthetic apparatus. This thesis elucidates the role and function of the enzyme magnesium chelatase, which catalyses the insertion of magnesium (II) into protoporphyrin IX and is the first committed enzyme of the chlorophyll biosynthetic pathway. Magnesium chelatase consists of three proteins, referred to as the I-, D- and H-subunits. In this thesis, the main focus has been to characterize the intermediate sized 70 kDa D-subunit. Previously, the crystal structure of R. capsulatus I-subunit was determined and electron microscopy analysis revealed that the protein formed a hexameric ring structure. The protein was found to belong to the AAA (ATPases associated with various cellular activities) family of proteins. Analysis of the amino acid sequence of the D-subunit suggests that the protein contain an atypical AAA-domain in the N-terminus and an integrin I domain in the C-terminus, which is linked by a proline rich and negatively charged region. In paper II, we present the structure of the R. capsulatus ID-complex of magnesium chelatase at 23 Å resolution by negative staining electron microscopy. The structure demonstrates that the proteins form a large complex of two hexameric rings. In paper I, we have characterized five recessive barley mutants defective in the D-subunit of magnesium chelatase and it was concluded that an unstable D-protein causes chlorophyll deficiency in vivo. The D-protein was further investigated in vitro by using heterologously expressed Rhodobacter capsulatus BchD. In vitro, inactive mutants exerted a dominant effect on magnesium chelatase activity, indicating that the D-proteins are cooperative and play an active role during catalysis. However, in paper III it was demonstrated that the dominant effect was dependant on the preparation of protein samples prior analysis. These observations lead to further investigations and discussions concerning the assembly and stability of the D-protein itself and in complex with the I-protein. We hypothesize that in the ID-complex, the D-protein form a stable hexameric platform for the unstable and ATPase active I-protein. A majority of the proteins involved in the photosynthetic apparatus are encoded by genes of the nuclear genome. Intermediates of the chlorophyll biosynthetic pathway have been found to function as signal molecules in chloroplast-to-nucleus signal transduction. In paper IV we investigated the signal transduction of barley mutants defective in magnesium chelatase and magnesium protoporphyrin IX monomethylester cyclase by using microarray technology.