Faculty
Schulich School of Medicine and Dentistry
Supervisor Name
Brian Shilton
Keywords
NQO2, NQO1, quinone reductase, cofactor specificity, NADH, enzyme kinetics, evolution, ancestral state reconstruction
Description
NQO1 and NQO2 are closely related quinone reductases, which are FAD-linked enzymes that catalyze the 2-electron reduction of quinones. Whereas NQO1 has a well-defined role in cellular detoxification of quinones, NQO2’s function is less clear; it uses the conventional cofactors NADH/NADPH inefficiently compared to smaller nicotinamide cofactors, which are often present in low cellular amounts. This unique cofactor specificity suggests that NQO2 may have non-enzymatic functions, such as a role in intracellular signalling.
The goals of this research project include: 1) examining whether cofactor specificity is conserved in amniote NQO2 enzymes and 2) re-constructing ancient enzymes along the evolutionary pathway of NQO1 and NQO2 to determine the molecular origins of cofactor specificity differences. The reductive half-reactions of H.sapiens, A.platyrhynchos, and A.mississippiensis NQO2 with NADH were investigated using stopped-flow apparatus. With increasing NADH concentration, the observed rate constant for NQO2 reduction for all three amniotes increased before reaching saturation. This indicates that the enzyme-substrate complex for NQO2 reduction exists in rapid equilibrium, leading to the conclusion of non-productive NADH binding across all amniote NQO2s. To examine the evolution of NQO1 and NQO2, a phylogenetic tree was created using amino acid sequences of numerous species. While the characterization of ancient enzyme kinetics is still ongoing, the successful expression of various ancestral proteins confirmed our analysis of the phylogeny and predicted sequences.
Acknowledgements
Dr. Brian Shilton, Faiza Islam, Western Research, Western Libraries, Student Experience
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Document Type
Poster
Included in
The Cofactor Specificity of NQO2
NQO1 and NQO2 are closely related quinone reductases, which are FAD-linked enzymes that catalyze the 2-electron reduction of quinones. Whereas NQO1 has a well-defined role in cellular detoxification of quinones, NQO2’s function is less clear; it uses the conventional cofactors NADH/NADPH inefficiently compared to smaller nicotinamide cofactors, which are often present in low cellular amounts. This unique cofactor specificity suggests that NQO2 may have non-enzymatic functions, such as a role in intracellular signalling.
The goals of this research project include: 1) examining whether cofactor specificity is conserved in amniote NQO2 enzymes and 2) re-constructing ancient enzymes along the evolutionary pathway of NQO1 and NQO2 to determine the molecular origins of cofactor specificity differences. The reductive half-reactions of H.sapiens, A.platyrhynchos, and A.mississippiensis NQO2 with NADH were investigated using stopped-flow apparatus. With increasing NADH concentration, the observed rate constant for NQO2 reduction for all three amniotes increased before reaching saturation. This indicates that the enzyme-substrate complex for NQO2 reduction exists in rapid equilibrium, leading to the conclusion of non-productive NADH binding across all amniote NQO2s. To examine the evolution of NQO1 and NQO2, a phylogenetic tree was created using amino acid sequences of numerous species. While the characterization of ancient enzyme kinetics is still ongoing, the successful expression of various ancestral proteins confirmed our analysis of the phylogeny and predicted sequences.