Characterisation of three cofactor-containing proteins from the oral bacterium Fusobacterium nucleatum

Fusobacterium nucleatum, a Gram-negative oral bacterium, is linked to periodontal disease, colorectal cancer and adverse pregnancy outcomes. The research herein uncovers the kinetic, structural and mechanistic properties of three F. nucleatum proteins that are potential therapeutic targets. Chapter 2 focuses on the characterisation of flavodoxin (FnFld), an FMN-containing electron transfer protein. FnFld was shown to have the highest recorded reduction potentials amongst characterised long-chain flavodoxins. Fluorescence binding studies revealed that the elevated potentials are due to preferred binding of the FMN hydroquinone anion over the oxidised FMN quinone. The structural rationale for the electropositive reduction potentials was provided through the x-ray crystal structure of FnFld. Chapter 3 investigates the thermodynamic and biophysical effects of four naturally occurring substitutions present in FnFld. A glycine to lysine substitution in a highly conserved phosphate-binding loop does not elicit a dramatic effect on FMN binding or the redox properties of the flavin cofactor, but it does contribute to a positively charged binding cleft that may serve as a redox partner recognition site. Three other residues, Y56, N89 and F93 were shown to modulate the electropositive reduction potentials. Chapter 4 investigates lanthionine synthase (LS), which belongs to the fold II family of PLP-dependent enzymes. Although LS is catalytically promiscuous like other family members, it shows catalytic preference toward the condensation of two molecules of L-cysteine to produce lanthionine and H₂S. Inspection of the LS crystal structure revealed a novel loop region containing S234 that forms electrostatic interactions with lanthionine. The catalytic importance of this residue was confirmed through mutagenesis. Chapter 5 focuses on the characterisation of a bifunctional ornithine decarboxylase/arginase (ODA). Sequence alignment revealed that ODA contains two naturally occurring amino acid substitutions in the conserved arginase binuclear Mn²+ binding site. ICP-MS analysis showed that wild type ODA is devoid of Mn²+ . Restoration to the conserved Mn²+ binding site improved Mn²+ binding, but not catalytic activity. We confirmed that H119, D199 and K230 are vital residues in the ornithine decarboxylase active site. Finally, known inhibitors of ornithine decarboxylase and arginase had no effect on ODA activity, suggesting that the enzyme contains distinct active sites.Science, Irving K. Barber Faculty of (Okanagan)Graduat