Abstract
Bacteriochlorophyll (BChl) c molecule, a main light-harvesting pigment in green photosynthetic bacteria, has a methyl group at the C-20 position of chlorin π-system. It is found that the C-20 methylation is catalyzed by a methyltransferase BchU in BChl c biosynthesis pathway, using S-adenosyl-L-methionine as a methyl group donor. We have determined the 3D structure of BchU by the X-ray crystallographic study. However, a chlorin-binding site was not fully resolved due to the low occupancy of an artificial chlorin substrate bound to it. Here, we report the in vitro analysis of BchU activity to investigate the recognition mechanism of chlorin substrates. BchU from Chlorobaculum tepidum was overexpressed in E. coli as a His-tagged protein and purified. BchU assay using various synthetic chlorophyllide (Chlide) derivatives indicated that BchU-catalyzed C-20 methylation would occur only in the branch of the biosynthestic pathway of BChl c after a partial D-ring reduction of protoChlide a and a subsequent C-132 demethoxycarbonylation of Chlide a. We will also report kinetic analysis of BchU and discuss a molecular structure of its chlorin-binding site.
