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

Hydrophobicity controls many aspects of protein and enzyme function. Although hydrophobic tuning can be somewhat achieved with canonical amino acids, the incorporation of non-canonical amino acids further extends this ability to enable new and improved functionality. Here 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. As a proof of concept, we demonstrate the utility of hydrophobic tuning based on non-canonical amino acids (ncAAs) to engineer a bacterial laccase, which is both a classical metalloenzyme and a high-value catalyst for industrial processes. The resulting mutations substantially improved the catalytic activity, particularly the turnover frequency and total turnover number. To understand this improved functionality, the redox potentials, electronic spectra and structure-function relationships were examined. Combining traditional directed evolution with ncAA-based engineering resulted in further improvements in catalysis, which were contextualized by analysing the changes imparted from these two methods.