Enzymology of cofactor and amino acid metabolism in anaerobic bacteria

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Over the past decade, my NSERC-funded research program has provided critical insight into mechanisms mediated by coenzyme containing enzymes, for example coenzyme-B12 radical chemistry, flavin-mediated electron transfer and monooxygenations and ß-replacement reactions catalyzed by PLP. Over the next five years, I will focus my program on enzymes and accessory proteins that function in heme, B12 and cationic amino acid metabolism in the phyla of Fusobacteria. Anaerobic lysine fermentation has been shown to control interspecies interactions mediated by the oral bacterium Fusobacterium nucleatum as well as the proliferation and virulence of the veterinary pathogen, Fusobacterium necrophorum. Our first objective is to characterize the two remaining unannotated gene products in the lysine fermentation pathway. I hypothesize that these proteins serve as novel metallochaperones that function to reactivate a coenzyme B12 enzyme, which catalyzes the second step in the fermentation pathway. This line of inquiry builds upon my existing research strength and NSERC- funded research program in radical-mediated reactions catalyzed by coenzyme B12 aminomutases. It also aims to expand our knowledge of the mechanisms by which coenzyme B12-enzymes contend with adventitious damage to the metabolically expensive B12 cofactor. The second main objective of our research program is to elucidate the structure, function and physiological role of an ornithine decarboxylase/arginase from F. nucleatum. I hypothesize that this enzyme supports biofilm growth through production of the polyamine, putrescence. We aim to determine the structure of the enzyme and develop a complete functional characterization of the enzyme and its active variants. We also aim to determine the physiological role of the protein through gene disruption and complementation experiments. This line of inquiry will broaden our research expertise while expanding our knowledge of the role of this enzyme in development of biofilm. Our third objective is to functionally and structurally characterize a novel flavodoxin that has the capacity to bind heme. Based on its genomic location, I hypothesize that serves as both a reductant and heme chaperone in a radical-mediated iron acquisition pathway. This research project also builds upon my expertise in flavin-mediated electron transfer and radical-mediated enzymatic reactions. The research will also provide insight into how some prevalent anaerobic bacteria acquire iron in the oral cavity, for which little is currently known. Highly qualified personnel engaged in this research program will have the opportunity develop technical expertise in methods that span the disciplines of biophysics, biochemistry, genetics, microbiology and molecular biology. They will also have the opportunity to engage in collaborative research and critical analysis, preparing them for fulfilling careers in academia, industry and government.