Artificial self-sufficient P450 in reversed micelles

Abstract

Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration.

Figure 1

Reaction scheme for d-camphor hydroxylation by branched P450cam with cofactor regeneration in a reversed micellar system.

Figure 2

(a) Relationship between the initial rate of NADH oxidation and the concentration of branched P450cam at a water content (W₀) of 16.7. The reaction mixture contained 5 mM d-camphor and 80 μM NADH. (b) Effect of W₀ on the initial activities of branched P450cam (open circles) and an equimolar mixture of PdR, PdX and P450cam (closed circles). The reaction mixtures contained 0.05 μM protein, 5 mM d-camphor and 80 μM NADH. The reversed micelle solution containing PdR, PdX and P450cam was prepared by adding a mixture of PdR, PdX and P450cam to a starter reversed micelle solution (see experimental section).

Figure 3

Relationship between initial rate of NADH oxidation and the concentration of d-camphor at a W₀ of 17. The reaction mixture contained 0.05 μM branched P450cam and 80 μM NADH.

Figure 4

UV-Vis spectrum of 2 μM branched P450cam with 5 mM d-camphor in the reversed micellar system at a W₀ of 17.

Figure 5

(a) Relationship between the initial activity and the concentration of A. pernix ADH with 70 μM NAD⁺ at a W₀ of 16.7. (b) Effect of W₀ on the initial activity of A. pernix ADH at 1.2 μM.

Figure 6

Relationship between the initial reaction rate of A. pernix ADH and the concentration of 2-pentanol in 50 mM potassium phosphate buffer, pH 7.4, containing 150 mM KCl. The reaction mixture contained 1.2 μM A. pernix ADH and 70 μM NAD⁺.

Figure 7

(a) The consumption of d-camphor by branched P450cam (1 μM) in the reversed micellar system after 24 h in the presence (37 μM) and absence of A. pernix ADH with 1 mM NADH and at a W₀ of 16.7. (b) Effect of W₀ on the consumption of d-camphor after 24 h in the coupled reaction containing 1 μM branched P450cam and 37 μM A. pernix ADH with 1 mM NADH. (c) Effect of d-camphor concentration on the consumption of d-camphor after 24 h in the coupled reaction containing 1 mM NADH and at a W₀ of 16.7. (d) Effect of initial NADH concentration on the consumption of d-camphor (open circles) and on the TTN of NADH (closed circles) after 24 h in the coupled reaction containing 20 mM d-camphor and at a W₀ of 16.7.

Figure 7

(a) The consumption of d-camphor by branched P450cam (1 μM) in the reversed micellar system after 24 h in the presence (37 μM) and absence of A. pernix ADH with 1 mM NADH and at a W₀ of 16.7. (b) Effect of W₀ on the consumption of d-camphor after 24 h in the coupled reaction containing 1 μM branched P450cam and 37 μM A. pernix ADH with 1 mM NADH. (c) Effect of d-camphor concentration on the consumption of d-camphor after 24 h in the coupled reaction containing 1 mM NADH and at a W₀ of 16.7. (d) Effect of initial NADH concentration on the consumption of d-camphor (open circles) and on the TTN of NADH (closed circles) after 24 h in the coupled reaction containing 20 mM d-camphor and at a W₀ of 16.7.

Figure 8

(a) Time course of d-camphor consumption by branched P450cam (open circles) and by an equimolar mixture of P450cam, PdX and PdR (closed circles) in the presence of A. pernix ADH at a W₀ of 16.7. (b) The stability of branched P450cam (open symbols) and of A. pernix ADH (closed symbols) in the reversed micellar system at a W₀ of 16.7 (circles) and in an aqueous solution (squares).