Are Heme-Dependent Enzymes Always Using a Redox Mechanism? A Theoretical Study of the Kemp Elimination Catalyzed by a Promiscuous Aldoxime Dehydratase

Abstract

The design of biocatalysts is a goal to improve the rate, selectivity and environmental friendship of chemical processes in biotechnology. In this regard, the use of computational techniques has provided valuable assistance in the design of enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM simulations have allowed getting an insight into the mechanism of a promiscuous aldoxime dehydratase (OxdA) for the Kemp elimination. We first demonstrate that, based on the use of linear response approximation (LRA) methods, the lowest energy electronic state of the benzisoxazole placed in the active sit of OxdA corresponds to a singlet state, being the triplet and the quintet state higher in energy. The presence of a heme group in the active site of the OxdA promiscuous enzyme opens the possibility of exploring a redox mechanism, similar to the one proposed in other reactions catalysed by heme-dependent enzymes. In addition, according to the geometrical analysis of the active site of this aldoxime dehydratase, the presence of a good base in the active site, His320, the proper pose of the substrate assisted by the porphyrin and an adequate electrostatic environment to stabilize the negative charge developed in the oxygen leaving group, makes available an acid/base mechanism. Comparison of the results derived from the exploration of both acid/base and redox mechanisms at B3LYP(Def2-TZVP)/MM level, shows how the later render the most favourable reaction path within the quintet state. The obtained activation free energy is in good agreement with the activation energy that can be deduced from the experimentally measured rate constant.