Abstract Knezevic, Neven

Fusobacterium nulceatum is a common oral bacterium that is indirectly implicated in periodontal

disease by providing an anaerobic environment within dental plaque for pathogenic bacteria such
as Porphyromonas gingivalis to thrive. Fusobacterium nucleatum contains a unique bifunctional
enzyme called ornithine decarboxylase arginase (ODA). The enzyme produces the polyamine,
putracine essential for the formation of the biofilm associated with dental plaque.
Characterization of ODA is an important first step towards design of a successful inhibitor for
the treatment of periodontal disease. The gene for ODA was successfully amplified from F.
nulceatum genomic DNA using the polymerase chain reaction. The coding sequence was
subsequently cloned into an E. coli expression vector for expression in the E. coli strain,
Rossetta(DE3) pLysS. The recombinant enzyme was purified by affinity chromatography. The
arginase active site catalyzes the hydrolysis of arginine to form ornithine and urea. Ornithine
decarboxylase converts ornithine to putrescine and carbon dioxide. UV-visible spectroscopic
assays confirmed that L-ornithine but not D-ornithine can bind to the ornithine decarboxylase
active site. Ornithine decarboxylase catalytic site activity was explored by performing steadystate kinetic coupled assay. Unfortunately, background levels of CO2 present in buffer prevented
accurate determination of catalytic turnover. However, a coupled assay was able to measure
catalytic activity of the arginase active site.