Engineering and assaying of cytochrome P450 biocatalysts

Abstract

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.

Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors

“…Until 2008, as much as 7,232 single P450 enzymes have been identified [6]. The amino acid sequence of the peptide chain can be quite diverse.…”

“…As early as 1978, the first P450-based enzyme electrode was presented [4], which was followed by a whole spectrum of different P450-based sensors for the quantification of drugs, metabolites (cholesterol) or pesticides [5][6][7][8][9]. The efficiency of electrode coupling of the biocatalytic systems is the crucial step in the development of drug sensors.…”

Activation of Molecular Oxygen in Cytochromes P450

“…This change in the coordination state of the heme iron gives rise to an upshift of redox potential, which is essential for efficient reduction of the enzyme from the ferric to the ferrous state The difference in redox potentials between pentacoordinated and hexacoordinated porphyrins can be illustrated using the thermodynamic cycle shown in Fig 33: Here the ligand L can bind to the heme iron with binding constants K 1 and K 3 , which are different for the ferric and ferrous states, while K 2 and K 4 define the redox equilibria for the fivecoordinated high-spin and six-coordinated lowspin heme iron respectively [16] The overall redox equilibrium between Fe 3 + and Fe 2 + , ie, the midpoint potential, can be shifted towards the strong binder, if the ligand is present [34] In the aqueous solution, water or a hydroxide always favors the ferric state as compared to ferrous, so that K 1 > 1 and K 3 < 1, and Fe 3 + is typically sixcoordinated in cytochromes P450 in the absence of a substrate, while Fe 2 + is five-coordinated As a result, the thermodynamic (redox) equilibrium between the ferric and ferrous states of the heme iron is shifted to the former in the absence of substrates, while substrate binding displaces the water molecule from the sixth coordination position, thus destabilizing the ferric state and lifting the midpoint potential Experimentally measured shifts of the redox potentials in cytochromes P450 caused by substrate binding are in the range 80- [50] Such results are probably due to the extremely low solubility of cholesterol and its derivatives and slow redistribution between the aqueous phase and lipid bilayers [19] The kinetics of NAD(P)H-dependent reduction of cytochromes P450 in the presence of their redox partners almost always strongly depends on the presence of their substrates Exceptions from this general rule are reported for several cytochromes P450 that are predominantly in the high-spin ferric state even before addition of a substrate, such as CYP1A2 [51][52][53] These observations are in line with the redox thermodynamics modulated by substrate binding described above (Fig 33) Typically, reduction of substrate-free P450 enzymes is very slow with apparent rates in the range of 10 − 4 -10 − 2 s − 1 [54], and is much faster (sometimes by several orders of magnitude) in the substrate-bound state [17] Sometimes the first electron-transfer step is identified as the rate-limiting step, as shown for CYP7A1 [55] The significant acceleration of P450 reduction in the presence of substrates is easily seen in the steady-state kinetics of NAD(P) H consumption, as reported for both bacterial and eukaryotic cytochromes [56] The acceleration of NAD(P)H oxidation can be used as an empirical test for the screening of new compounds as potential substrates for a given cytochrome P450 [57,58] or as a rough measure of P450 activity [59] Interactions with redox partners are not only necessary to bring the electron donor close to the heme for efficient electron transfer Recent structural studies of the complex of CYP101A1 with its natur...…”

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

“…[4] A longsought and challenging goal in cytochrome P450 research is to capitalize on the selective introduction of molecular oxygen into double bonds, at allylic positions, and also into non-activated CÀH bonds for the production of fine chemicals and pharmaceuticals that are difficult to prepare by traditional organic synthesis. [5] Prokaryotic P450s are soluble and therefore easy to purify and to handle, unlike their eukaryotic counterparts, which are, with some exceptions, integral membrane proteins. [3] However, as external monooxygenases, prokaryotic P450s in general need to interact with their soluble electron donor partners to display functional activity.…”