A redox-mediated Kemp eliminase

Abstract

The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N-O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)-N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.

Figure 1. Mechanisms of two different types of Kemp eliminases.

(a) Base-mediated exothermic E2 elimination. (b) A possible redox-mediated process.

Figure 2. Overview of substrate 1 accommodated in the active site of WT P450-BM3.

The representative snapshot was taken from molecular dynamic (MD) simulation, revealing substrate 1 in the binding pocket of P450-BM3 and the surrounding residues. As can be seen, there are no residues which could act as in an acid/base mechanism.

Figure 3. Redox-mediated Kemp elimination of substrate 1.

(a) A representative snapshot in the equilibrium molecular dynamic (MD) trajectory showing the active site structure of WT P450-BM3; note that the nitrogen atom of substrate 1 is directed toward Fe(II), with an average Fe—N1 distance of 4.44 Å. (b) Quantum mechanics/molecular mechanics (QM/MM) (UB3LYP/B2) relative energies (kcal mol⁻¹) for the redox-mediated Kemp elimination of 1. All values are dispersion-corrected. The values in parentheses also include zero-point energy (ZPE) corrections. For clarity, the reactivity energy profile in the singlet state is provided in Supplementary Fig. 6. Cartesian coordinates of QM region for all species from QM/MM calculations are available as Supplementary Data 1. (c) A representative snapshot in the equilibrium MD trajectory showing the active site structure of variant F87G, with an average Fe—N1 distance of 4.11 Å. (d) Catalytic cycle for redox-mediated Kemp elimination.

Figure 4. Metabolism of leflnomide with human P450.

Human P450 catalyses the isoxazole ring scission of leflunomide, an immunomodulatory therapeutic drug, leading to teriflunomide (A771726).

Figure 5. Activity test of P450-BM3 variants in metabolizing leflunomide.

Reaction conditions: 500 μl reaction consisting of 1 μM enzyme, 500 μM substrate and 1 mM NADPH in phosphate buffer (50 mM, pH 8.0, 100 mM NaCl), 25 °C, 1,000 r.p.m. for 1 h. All reactions were performed in triplicate, and error bars show ±s.d.