COBALAMIN (VITAMIN-B-12) BIOSYNTHESIS - CLONING, EXPRESSION AND CRYSTALLIZATION OF THE BACILLUS-MEGATERIUM S-ADENOSYL-L-METHIONINE-DEPENDENT COBALT-PRECORRIN-4 TRANSMETHYLASE CBIF

The Bacillus megaterium cbiF, encoding the cobalt-precorrin-4 S-adenos yl-L-methionine-dependent transmethylase of the anaerobic cobalamin bi osynthetic pathway, has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The protein was purified to h omogeneity by a combination of metal chelate chromatography and high-r esolution anion-exchange chromatography. The protein migrated with a s ubunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant p rotein is a homodimer. The His-tagged protein was physiologically acti ve as it was able to complement a Salmonella typhimurium cbiF mutant. However, the protein did not bind S-adenosyl-L-methionine with the sam e avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identifica tion of two discrete crystal forms. One of these forms has been charac terised and a full data set collected.