Electronic Thesis and Dissertation Repository


Master of Science




Dr. Brian Shilton


Quinone Reductase 2 (NQO2) is a part of an enzyme family implicated in detoxification of the cell. This enzyme differs from its highly similar sister protein Quinone Reductase 1 (NQO1) by its unique cofactor preference for dihydro-nicotinamide riboside (NRH) instead of NADH. Cellular levels of NRH have not been characterized, contributing to the mystery surrounding the physiological role of NQO2. This project focused on identifying what determines the cofactor specificity of NQO2, and engineering it to use NADH instead of NRH. The investigation included optimization of protein purification, an in silico analysis that revealed the different stereochemistry of NQO2/1 from other flavoenzymes, and an x-ray analysis of NQO2 co-crystallized with NADH. An informed site-directed mutagenesis approach yielded a 70-fold increase in the catalytic efficiency of NQO2 utilizing NADH, compared to the wildtype. The results of this project help characterize the cofactor preference of NQO2 on a molecular level.

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