Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
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This project was supported by the European Research Council (ERC starting grant no. 280010) and the Netherlands Organisation for Scientific Research (NWO) (Vici grant 724.013.003, and Veni grant 722.017.007). G.R. acknowledges support from the Ministry of Education Culture and Science (Gravitation programme no. 024.001.035). C.M. acknowledges a Marie Skłodowska Curie Individual Fellowship (project no. 751509). The authors thank A. Borg for preparing the plasmid harbouring the bcPadR1 gene and M. de Vries for performing trypsin digestion and LC–MS/MS analyses.
The authors declare no competing interests.
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Supplementary Tables 1–3, Supplementary Figures 1–8, a list of materials and equipment used, procedures for the chemical syntheses of substrates and products, an extended Methods section, a list of primers used, NMR spectra of synthesized compounds, and Supplementary References
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Drienovská, I., Mayer, C., Dulson, C. et al. A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue. Nature Chem 10, 946–952 (2018). https://doi.org/10.1038/s41557-018-0082-z
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