(582cx) Introducing Nitroreductase Functionality in Ene Reductases

Park, J. T. - Presenter, Georgia Institute of Technology
Gómez Ramos, L. M., Georgia Institute of Technology
Bommarius, A., Georgia Institute of Technology

Nitroreductases are enzymes that reduce nitro compounds to corresponding nitroso, and hydroxylamino compounds. In the past 20 years, they have gathered interest in several applications such as degradation of xenobiotic nitroaromatics, and activation of prodrugs in medical applications. However, the use of nitroreductases in these fields is limited due to the lack of thermal and kinetic stability, which is shown by their low melting temperatures and high deactivation constants.

To overcome this limitation, we created a nitroreductase from a stable ene reductase scaffold through protein engineering methods such as rational design, and site-saturation mutagenesis. In this study, we characterize nitroreductases from Salmonella typhimurium, Enterobacter cloacae, and Mycobacterium smegmatis, to identify residues significant for catalysis and substrate binding. With this knowledge, mutations are performed to engineer nitro reduction functionality from ene reductases, namely xenobiotic reductase A (XenA) from Pseudomonas putida, YersER from Yersinia bercoviei and KYE1 from Kluyveromyces lactis. The novel enzymes are characterized with steady-state kinetics and conversion studies.