Probing structure of heme A synthase from Bacillus subtilis by site-directed mutagenesis.
Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His(6)-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K.K., and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287-295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild-type level. On the basis of our findings, we proposed a helical-wheel-projection model of CtaA.
Mogi
Tatsushi
T
Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Tokyo, Japan. tmogi@m.u-tokyo.ac.jp
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D006639:Histidine; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification; D009842:Oligopeptides; D010084:Oxidation-Reduction; D011485:Protein Binding; D013057:Spectrum Analysis
D001426:Bacterial Proteins; C090098:CtaA protein, bacteria; D003573:Cytochrome b Group; C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D050505:Mutant Proteins; D009842:Oligopeptides; C069808:heme O; C027728:heme a; D006418:Heme; D006639:Histidine