MetREx: a protein design approach for the exploration of sequence-reactivity relationships in metalloenzymes

Metalloenzymes represent a particular challenge for any rational (re)design approach because the modeling of reaction events at their metallic cofactors requires time-consuming quantum mechanical calculations, which cannot easily be reconciled with the fast, knowledge-based approaches commonly applied in protein design studies. Here, an approach for the exploration of sequence-reactivity relationships in metalloenzymes is presented (MetREx) that consists of force field-based screening of mutants that lie energetically between a wild-type sequence and the global minimum energy conformation and which should, therefore, be compatible with a given protein fold. Mutant candidates are subsequently evaluated with a fast and approximate quantum mechanical/molecular mechanical-like procedure that models the influence of the protein environment on the active site by taking partial charges and van der Waals repulsions into account. The feasibility of the procedure is demonstrated for the active site of [FeFe] hydrogenase from Desulfovibrio desulfuricans. The method described allows for the identification of mutants with altered properties, such as inhibitor-coordination energies, and the understanding of the robustness of enzymatic reaction steps with respect to variations in sequence space.