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Free keywords:
Kemp elemination; Redox mechanism; P450 monooxygenase; Quantum mechanical/ molecular mechanical; X-ray structure; Saturation mutagenesis
Abstract:
For three decades the biocatalytic version of the Kemp elimination of 5‐nitro-benzisoxazole (1) has
served as a forum for testing the creation of different artificial enzymes, the primary aim being to reveal mechanistic intricacies and to extend our understanding of enzymatic catalysis as such. In general, acid/base catalysis pertains, but recently a novel redox based mechanism was postulated
when using P450‐BM3 mutants as scaffolds. In the present study, we report an surprising discovery made upon employing new P450‐BM3 variants generated by rational enzyme design, which points to the existence of a new and different redox based mechanism. X‐ray structural data and theoretical analyses based on MD simulations and QM/MM calculations support this conclusion. The results of this study are of relevance in the human metabolism of therapeutic drugs and in redox mediated biosynthesis catalyzed by P450s.
