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.
Mogi
Tatsushi
T
Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Tokyo, Japan. tmogi@m.u-tokyo.ac.jp
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli; D006418:Heme / Q000096:biosynthesis / Q000737:chemistry; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D013057:Spectrum Analysis
D001426:Bacterial Proteins; C069808:heme O; D006418:Heme; D006639:Histidine; D019883:Alkyl and Aryl Transferases; C078356:CyoE protein, Bacteria