CAREER: Molecular Evolution of Oxidant Specificity in Bacterial Flavoprotein Amine Oxidases

With this CAREER award, supported by the Chemistry of Life Processes Program in the Division of Chemistry, Frederick Stull from Western Michigan University is studying the evolution of oxidant specificity in bacterial flavoprotein amine oxidases. Flavoprotein amine oxidases are enzymes that catalyze critical reactions throughout cellular metabolism, and most are thought to transfer electrons from their organic substrates to molecular oxygen. It was recently discovered that certain bacterial flavoprotein amine oxidase enzymes break this mold by transferring electrons from their substrates to cytochrome c proteins instead of directly to molecular oxygen. This project is evaluating if this use of cytochrome c is more widespread among bacterial flavoprotein amine oxidases than previously realized and seeks to understand how these divergent functions evolved in this widespread enzyme family. The basic science knowledge obtained from this project could be used to achieve reactions that are useful for biotechnology. The graduate and undergraduate students involved in this project will gain hands-on experience in spectroscopy, transient kinetics and enzyme engineering. Selected enzymes from this project are to be used to develop a hypothesis-driven laboratory course for undergraduate students that directly contributes to the scientific goals of this project. This project will also include development of a platform for facilitating the engagement of undergraduate work-study recipients in hands-on research. Frederick Stull and his team have recently discovered that nicotine oxidoreductase and pseudooxynicotine amine oxidase, both members of the flavoprotein amine oxidase (FAO) superfamily, are not actually oxidases. These two enzymes transfer electrons from their substrates to a cytochrome c protein instead of O2, meaning that they are dehydrogenases. This project will test the hypothesis that many other bacterial flavoprotein amine oxidases also use cytochrome c as an oxidant instead of O2, severely undermining the oxidase paradigm ascribed to the FAO enzyme family. The oxidant specificity of select bacterial FAOs that are predicted to be cytochrome c-utilizing dehydrogenases, as well as closely related oxidases, will be characterized by transient kinetics. Experimental evolution, phylogenetic analysis and ancestral reconstruction will be used to identify the structural features that dictate flavin-oxidant reactivity in bacterial FAOs. Information from this study will likely provide new insight into how flavoenzymes control the reactivity of their flavin cofactors. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.