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Bioconjugation and Active Site Design of Enzymes Using Non-natural Amino Acids
- Kwon, Inchan, Yang, Byungseop
- Industrial & engineering chemistry process design and development 2017 v.56 no.23 pp. 6535-6547
- active sites, amino acids, binding capacity, biocatalysts, biotin, chemical bonding, chemical reactions, dihydrofolate reductase, folic acid, formate dehydrogenase, mannitol, methotrexate, mice, process design, protein engineering, substrate specificity, value-added products
- Enzymes are biocatalysts that play key roles in diverse chemical reactions in living organisms and industrial conversion processes generating value-added products. Since wild-type enzymes obtained from nature are normally not optimal for various applications, enzyme engineering is usually required for enhanced or new properties. Site-specific incorporation of a non-natural amino acid became a powerful protein-engineering tool. In this short review, we briefly summarize our contribution to enzyme complex formation and active site design of enzymes using the technique of site-specific incorporation of a non-natural amino acid. First, site-specific incorporation of a non-natural amino acid at a permissive site of an enzyme led to bioconjugation to other molecules without compromising critical properties. Murine dihydrofolate reductase (mDHFR) was site-specifically conjugated to a biotin via click chemistry to achieve site-specific immobilization. Similarly, formate dehydrogenase (FDH) was site-specifically conjugated to an organometallic catalyst for cofactor regeneration. Furthermore, successive applications of two different click chemistries allowed site-specific coupling of FDH and mannitol dehydrogenase for the enhanced overall reaction efficiencies via substrate channeling effects and active site-orientation control. Besides enzyme bioconjugation, precise control of non-natural amino acid incorporation also allows for active site modification with a non-natural amino acid. Introduction of a bulky non-natural amino acid into the mDHFR active site lowered binding affinity to its inhibitor methotrexate without compromising binding affinity to its substrate dihydrofolate. Similarly, introduction of a bulky non-natural amino acid into the mDHFR active site led to the alteration of substrate specificity toward a poor substrate folate over a good substrate dihydrofolate.