title: Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome p450 redox partner.
abstract: Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6<pH<8; this enabled the quinone/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) redox potentials to be determined. Our results show that cindoxin might be somewhat different to other flavodoxins that interact with P450s, in which generally only one couple is important. Both redox states of cindoxin could be catalytically relevant. A catalytically active system was reconstituted in vitro with E. coli flavodoxin reductase (Fpr) acting as the terminal redox partner in the absence of CdR. Our results show that Cdx and Fpr support regio- and stereoselective P450(cin)-catalysed cineole oxidation to (1R)-6beta-hydroxycineole with turnover rates up to 1500 min(-1). This system is tightly coupled with 80 % of NADPH reducing equivalents funnelled into substrate oxidation.
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia.
authors.forename: David B
authors.initials: DB
authors.lastname: Hawkes
authors.forename: Kate E
authors.initials: KE
authors.lastname: Slessor
authors.forename: Paul V
authors.initials: PV
authors.lastname: Bernhardt
authors.forename: James J
authors.initials: JJ
authors.lastname: De Voss
mesh_terms: D002954:Citrobacter / Q000201:enzymology*; D003001:Cloning, Molecular; D003511:Cyclohexanols / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D000077591:Eucalyptol; D005486:Flavin Mononucleotide / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D039821:Monoterpenes / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D003511:Cyclohexanols; D005418:Flavodoxin; D039821:Monoterpenes; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D000077591:Eucalyptol