Hydrophobicity controls many aspects of protein and enzyme function.^[1-3] Although hydrophobic tuning can be achieved to a limited extent with canonical amino acids, the incorporation of non-canonical amino acids (ncAAs) has the potential to further extend this ability, enabling new and improved functionality. We engineered a robust aminoacyl-tRNA synthetase/tRNA pair for the incorporation of hydrophobic amino acids, specifically cycloalkylalanines, in E. coli. These ncAAs were utilized to engineer the primary coordination sphere of bacterial laccase, a prototypical metalloenzyme with T1 copper site. Laccases are high value catalysts that can perform one-electron oxidation chemistry to catalyze a variety of transformations, including lignin polymerization/depolymerization and degradation of pollutants. Our ncAA-engineered mutants revealed enhanced redox potentials and catalytic properties, particularly for the oxidation of challenging phenolic substrates. Additionally, we demonstrated that further engineering of the second coordination sphere can result in an additional increase in catalytic activity, illustrating that these new tools can be combined with traditional directed evolution techniques.

[1] D. A. Estell, T. P. Graycar, J. V. Miller, D. B. Powers, J. A. Wells, J. P. Burnier, P. G. NG, Science 1986, 233, 659–663.

[2] J. A. Hunt, M. Ahmed, C. A. Fierke, Biochemistry, 1999, 38, 9054-9062.

[3] D. K. Garner, M. D. Vaughan, H. J. Hwang, M. G. Savelieff, S. M. Berry, J. F. Honek, Y. Lu, J. Am. Chem. Soc. 2006, 128, 15608-15617.