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Bioconjugation and Active Site Design of Enzymes Using Non-natural Amino Acids
Kwon, Inchan,Yang, Byungseop American Chemical Society 2017 INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH - Vol.56 No.23
<P>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.</P>
Thermostable and Long-circulating Albumin-conjugated Urate Oxidase for Gout Treatment
Na Hyun KWON,Byungseop YANG,Inchan KWON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
Urate oxidase from Aspergillus flavus provides effective therapy for gout. However, its clinical application faces several issues, including low thermostability and short circulation time in vivo. The recent study showed that urate oxidase derived from Arthrobacter globiformis (AgUox) exhibits high thermostability. Hence, to develop a thermostable and long-circulating therapeutics, we site-specifically conjugated human serum albumin (HSA) to AgUox via non-natural amino acid (frTet) incorporation and an inverse electron demand Diels-Alder (IEDDA) reaction. Among 14 candidates predicted, AgUox incorporating frTet at position 196 (Ag12) showed enzyme activity and thermostability comparable to wild-type AgUox. The IEDDA reaction led to high conjugation yield, and resulting Ag12-HSA retained enzyme activity. Furthermore, the Ag12-HSA had a serum half-life of 29 h in mice, roughly 17-times of that of wild-type AgUox. The results demonstrated conjugated AgUox as a promising therapeutic candidate for the treatment of related diseases.