Publications

• Selectivity in a barren landscape: the P450(BioI)-ACP complex.

  Cryle M.J.

  The cytochromes P450 (P450s) are a superfamily of oxidative haemoproteins that are capable of catalysing a vast range of oxidative transformations, including the oxidation of unactivated alkanes, often with high stereo- and regio-selectivity. Fatty acid hydroxylation by P450s is widespread across ... >> More

  The cytochromes P450 (P450s) are a superfamily of oxidative haemoproteins that are capable of catalysing a vast range of oxidative transformations, including the oxidation of unactivated alkanes, often with high stereo- and regio-selectivity. Fatty acid hydroxylation by P450s is widespread across both bacteria and higher organisms, with the sites of oxidation and specificity of oxidation varying from system to system. Several key examples are discussed in the present article, with the focus on P450(BioI) (CYP107H1), a biosynthetic P450 found in the biotin operon of Bacillus subtilis. The biosynthetic function of P450(BioI) is the formation of pimelic acid, a biotin precursor, via a multiple-step oxidative cleavage of long-chain fatty acids. P450(BioI) is a member of an important subgroup of P450s that accept their substrates not free in solution, but rather presented by a separate carrier protein. Structural characterization of the P450(BioI)-ACP (acyl-carrier protein) complex has recently been performed, which has revealed the basis for the oxidation of the centre of the fatty acid chain. The P450(BioI)-ACP structure is the first such P450-carrier protein complex to be characterized structurally, with important implications for other biosynthetically intriguing P450-carrier protein complexes. << Less

  Biochem Soc Trans 38:934-939(2010) [PubMed] [EuropePMC]

• Structural insights from a P450 Carrier Protein complex reveal how specificity is achieved in the P450(BioI) ACP complex.

  Cryle M.J., Schlichting I.

  Cytochrome P450(BioI) (CYP107H1) from the biotin operon of Bacillus subtilis forms a seven-carbon diacid through a multistep oxidative cleavage of a fatty acid linked to acyl carrier protein (ACP). Crystal structures of P450(BioI) in complex with three different length fatty acyl-ACP (Escherichia ... >> More

  Cytochrome P450(BioI) (CYP107H1) from the biotin operon of Bacillus subtilis forms a seven-carbon diacid through a multistep oxidative cleavage of a fatty acid linked to acyl carrier protein (ACP). Crystal structures of P450(BioI) in complex with three different length fatty acyl-ACP (Escherichia coli) ligands show that P450(BioI) binds the fatty acid such as to force the carbon chain into a U-shape above the active site heme. This positions the C7 and C8 carbons for oxidation, with a large additional cavity extending beyond the heme to accommodate the methyl termini of fatty acids beyond the site of cleavage. The structures explain the experimentally observed lack of stereo- and regiospecificity in the hydroxylation and cleavage of free fatty acids. The P450(BioI)-ACP complexes represent the only structurally characterized P450-carrier protein complexes to date, which has allowed the generation of a model of the interaction of the vancomycin biosynthetic P450 OxyB with its proposed carrier protein bound substrate. << Less

  Proc. Natl. Acad. Sci. U.S.A. 105:15696-15701(2008) [PubMed] [EuropePMC]

• Expression, purification, and characterization of BioI: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis.

  Stok J.E., De Voss J.

  Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI. We have subcloned biol and overexpressed the encoded protein, Biol. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxy ... >> More

  Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI. We have subcloned biol and overexpressed the encoded protein, Biol. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography. Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coli acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI 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 partners 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 isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to 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. << Less

  Arch. Biochem. Biophys. 384:351-360(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Carbon-carbon bond cleavage by cytochrome p450(BioI)(CYP107H1).

  Cryle M.J., De Voss J.J.

  Cytochrome p450(BioI)(CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis: we report here that the mechanistic pathway adopted by this multifunctional p450 for the in-chain cleavage of fatty acids is via consecutive formation of alcohol and threo-d ... >> More

  Cytochrome p450(BioI)(CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis: we report here that the mechanistic pathway adopted by this multifunctional p450 for the in-chain cleavage of fatty acids is via consecutive formation of alcohol and threo-diol intermediates, with the likely absolute configuration of the intermediates also reported. << Less

  Chem. Commun. (Camb.) 7:86-87(2004) [PubMed] [EuropePMC]