A heme-thiolate protein (P-450) which uses a flavodoxin-like redox partner to reduce the heme iron. Isolated from the bacterium Citrobacter braakii, which can use 1,8-cineole as the sole source of carbon.
a hydroxyl group on the substrate is vital, and in its absence catalytic turnover is effectively abolished. In the absence of the ethereal oxygen there is still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. The substrate itself is not important in controlling oxygen activation, but is essential for regio- and stereoselective substrate oxidation
redox partner is cindoxin, containing FMN. Cindoxin might be different to other flavodoxins that interact with P450s, as both redox states of cindoxin could be catalytically relevant. Cindoxin supports regio- and stereoselective P450cin-catalysed cineole oxidation to (1R)-6beta-hydroxycineole with turnover rates up to 1500 per min
the FAD/FMN reductase consists of two separate polypeptides where the FMN protein, cindoxin, shuttles electrons between the FAD-containing cindoxin reductase and P450cin. Reaction is highly ionic strength-dependent. The fully reduced hydroquinone is the electron-donating species. Surface interactions are rather different from other P450 proteins
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in complex with substrate 1,8-cineole, to 1.7 A resolution, and comparison with P450cam, EC 126.96.36.199. The active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s, is replaced by an ordered loop that results in substantial changes in active site topography. Cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe2S2 redox partner used by cytochrome P450cam
X-ray crystal structures of the substrate-free and -bound N242A mutant to 2.0 and 3.0 A resolution, respcetively. Mutation results in a reorientation of the substrate such that (R)-6'-hydroxycineole should be a major product
no change in characteristic CO-bound spectrum and spectrally determined KD for substrate binding. Mutation leads to modest effects on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation, but results in a reorientation of the substrate such that (R)-6'-hydroxycineole is a major product
significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 22% and 31%, respectively
significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 18% and 39%, respectively