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.

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…Cindoxin supports catalytic function of the cineole oxidizing P450cin (supplemental Fig. S4) (44). Flavodoxins typically have high affinity for their FMN cofactor (e.g.…”

Cytochrome P450 Is Present in Both Ferrous and Ferric Forms in the Resting State within Intact Escherichia coli and Hepatocytes

“…It was possible to augment the endogenous redox partners with the cognate electron donors for each P450 form: hCPR, for the human forms, cindoxin plus the surrogate endogenously expressed E. coli FdR for CYP176A1 (41), and putidaredoxin plus putidaredoxin reductase for CYP101A1. The effect of coexpressed electron donors on the P450 reduction state was most apparent for forms that showed less reduction by the endogenous electron donors, i.e.…”

P-LinK: A Method for Generating Multicomponent Cytochrome P450 Fusions with Variable Linker Length

“…As a reduction equivalent, NADPH is used in the electron transfer chain from CinB -> CinC -> CinA (P450cin). CinB is usually replaced in activity measurements by the E. coli NADPH-ferredoxin (flavodoxin) reductase (Fpr), which is, in contrast to CinB, highly expressed in its active form in E. coli (26, 27, 31). …”

“…In order to keep the systems comparable, an equimolar ratio was used instead of the optimal ratio of 1:8:2 for CinA, CinC, and Fpr (27). …”

“…Expression of E. coli flavodoxin reductase (Fpr) was performed as previously reported, and purification was performed with modifications to the protocol of Hawkes et al (27, 28). …”