Hydrophobic tuning with non-canonical amino acids in a copper metalloenzyme

Abstract

Hydrophobicity controls many aspects of protein and enzyme function. Although hydrophobic tuning can be achieved to a limited extent with canonical amino acids, the incorporation of non-canonical amino acids (ncAAs) further extends this ability to enable new and improved functionality. Herein, we engineer an aminoacyl-tRNA synthetase/tRNA pair for the site-specific genetic encoding of a set of bulky, hydrophobic amino acids, namely cyclopentylalanine, cyclohexylalanine, and cycloheptylalanine. With the resulting orthogonal translations systems, we demonstrate the utility of ncAA-based hydrophobic tuning to engineer a bacterial laccase, which is both a classical metalloenzyme and a high-value catalyst for industrial processes. The resulting mutations conveyed significant improvements in catalytic activity, particularly kcat and total turnover number. The redox potential and structure-function relationships were examined to elucidate the source of this improved functionality. We envision that these tools for hydrophobic tuning will be highly valuable for general enzyme engineering and also in other fields, including peptide chemistry.