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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.
