Abstract
The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous artificial enzyme that leverages the unique properties of a non-canonical amino acid (ncAA) para- aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, whilst the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semi-synthetic catalytic machinery. Our analysis provides valuable insights for the future engineering of effective artificial enzymes which employ either the widely used LmrR scaffold or pAF catalytic residue.
Supplementary materials
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Supporting Information
Description
Supporting Tables S1-S7 and Supporting Figures S1-S20, along with the Experimental and Computational
Methods
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