Over-expression and characterization of Bacillus subtilis heme O synthase.

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme O synthase in Bacillus subtilis (CtaB) has eight transmembrane helices and catalyses the transfer of a farnesyl group from farnesyl diphosphate to the 2-vinyl group on pyrrole ring A of ferrous heme B. In this study, we constructed the overproduction system for the B. subtilis CtaB in Escherichia coli. We isolated His(7)-CtaB by affinity chromatography and demonstrated the presence of the heme-binding site in heme O synthase. His(7)-CtaB binds substoichiometric amounts of heme B and O, substrate and unreleased product, respectively. Mutagenesis studies suggest that strictly conserved His199 present at the extra-cellular side of helix 5 would serve as the heme-binding site. We are hoping that the overproducing system for heme O synthase would help understanding of detailed mechanism on heme O biosynthesis and X-ray crystallographic studies.