The expression, purification and steady state analysis of magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803

Abstract

Magnesium protoporphyrin IX methyltransferase is an enzyme involved in the chlorophyll forming branch of tetrapyrrole biosynthesis. It catalyses the methylation of magnesium protoporphyrin (MgP) at the C6 propionic sidechain, to form magnesium protoporphyrin IX monomethylester (MgPME). The methyl donor for the reaction is S-adenosyl-L-methionine (SAM), which is converted to S-adenosyl-L-homocysteine (SAH). The chlM gene from the cyanobacterium Synechocystis PCC6803 was cloned into a pET9a-based expression plasmid and the methyltransferase was overproduced in E. coli. The resultant protein was purified to homogeneity using a nickel chelating column. A stopped assay has been developed using HPLC reversed phase chromatography to follow the evolution of MgPME in the steady state. This technique has been used to determine Vmax and Km values. The methyltransferase has also been titrated with both substrates, and binding was followed with a tryptophan fluorescence based assay. This method was used to obtain the Kd values for both substrates, to complement the other steady state parameters already determined.