Expression, purification, and characterization of biol: A carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has bee n proposed to involve a cytochrome P450 encoded by the gene biol. We have s ubcloned bioI and overexpressed the encoded protein, BioI. A purification p rotocol was developed utilizing ion exchange, gel filtration, and hydroxyap atite chromatography, Investigation of the purified BioI by UV-visible spec troscopy revealed spectral properties characteristic of a cytochrome P450 e nzyme. BioI copurifies with acylated Escherichia coil acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to B ioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox part ners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl -ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isol ated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 b inding most tightly to the enzyme. These fatty acids are also metabolized t o dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis . In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production. (C) 2000 Aca demic Press.