The ability to site-specifically incorporate a diverse set of unnatural amino acids (> 30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids ( UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the > 30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a > 2.3- fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.