(69a) Engineering Enzymes Using Nonnatural Amino Acids

Enzymes have become a very important player for modern biotechnlogoy.  Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties using protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, nonnatural amino acid incorporation (NAAI) has become a new and effective protein engineering technique. As an emerging application, we have explored whether NAAI can be used to improve enzyme properties. As a test protein, we chose murine dihydroxyfolate reductase (mDHFR) and incorporated several nonnatural amino acids in a site-specific manner. Site-specific incorporation of nonnatural amino acids into mDHFR did not perturb the correct folding of mDHFR. Properties of the mDHFR variants containing nonnatural amino acids (nonnatural mDHFR variants) including caatalytic activities, thermal stability, and resistance to inhibitors are being evaluated in order to obtain knowledges on how nonnatural amino acids modulate enzyme structure and properties. These findings would facilitate the validation of NAAI as a tool to engineer enzymes.