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Darbyshire, Amanda

University of British Columbia

Fusobacterium nucleatum is a gram-negative bacterium that is ubiquitous to the human oral cavity and has been associated to different pathologies. This thesis focuses on three distinct enzymatic pathways that are involved in F. nucleatum amino acid metabolism. Chapter 2 and 3 focus on serine synthase, a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible reaction of ʟ-cysteine and H₂O to form ʟ-serine and H₂S. The mechanism for this reaction requires an active site aspartic acid residue (D232) to act as a general acid/base catalyst. Hydrophobic residues surrounding D232 increase the basicity of the carboxylate side chain and restrict the range of nucleophilic substrates that can dock into the second binding site. Mutation of these aromatic residues allows for addition of thiol-based nucleophiles to the α-aminoacrylate intermediate of the PLP cofactor, thus allowing serine synthase to act as a potential biocatalytic tool for noncanonical amino acid synthesis. Chapter 4 focuses on lanthionine synthase, another PLP-dependent enzyme, which catalyzes the condensation reaction of ʟ-cysteine with a second equivalent of ʟ-cysteine to produce ʟ,ʟ-lanthionine and H₂S. In this chapter, we utilized the relaxed substrate scope of lanthionine synthase to produce different lanthionine analogs and test potential inhibitors. We also determined that the physiological production of meso-lanthionine in F. nucleatum is likely through the reaction of lanthionine synthase with ʟ-cysteine and D-cysteine and not through an epimerase. Fn1732 in the F. nucleatum genome may act as a racemase on an amino acid other than ʟ,ʟ- lanthionine. Chapter 5 investigates a putative metallochaperone system for lysine 5,6- aminomutase (5,6-LAM). 5,6-LAM is a PLP- and adenosylcobalamin (AdoCbl)-dependent enzyme involved in the second step of the lysine fermentation pathway, catalyzing the conversion of β-ʟ-lysine to erythro-3,5-diaminohexanoate. Like other AdoCbl-dependent enzymes, 5,6-LAM undergoes suicide inactivation during catalysis as a result of the loss of the 5′-deoxyadenosyl moiety and the irreversible oxidation of cob(II)alamin to hydroxocobalamin. We have identified kamB and kamC, two genes responsible for the ATP-dependent reactivation of 5,6-LAM. KamBC is structurally distinct from reactivating factors of other AdoCbl-dependent enzymes, and therefore adds to the list of proteins that have evolved to maintain cellular activity of AdoCbl-dependent enzymes.

Text

2024-05-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88331

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/