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 putida1, YersER from Yersinia bercoviei and KYE1 from Kluyveromyces lactis2.The novel enzymes show significant nitro reduction activity whereas the wild-type showed none. Successful first round single mutations were combined to determine additive and synergistic effects on reaction rates. The best variants were picked and characterized with steady-state kinetics and conversion studies. 1. Yanto, Y.; Yu, H. H.; Hall, M.; Bommarius, A. S., Characterization of xenobiotic reductase A (XenA): study of active site residues, substrate spectrum and stability. Chemical Communications 2010,  46 (46), 8809-8811.2. Yanto, Y.; Winkler, C. K.; Lohr, S.; Hall, M.; Faber, K.; Bommarius, A. S., Asymmetric Bioreduction of Alkenes Using Ene-Reductases YersER and KYE1 and Effects of Organic Solvents. Organic Letters 2011,  13 (10), 2540-2543.

Introducing nitroreductase functionality into ene reductases

247th National Meeting (2014)

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