INITIAL FUNCTIONAL CHARACTERIZATION OF BFO 2291 AND BFO 2294 FROM Tannerella forsythia; DEGRADERS OF CHONDROITIN SULFATE A

Periodontitis is characterized by the inflammation of the periodontal tissues in response to bacterial action. The Global Burden of Disease (GBD) 2010 study shows that periodontitis ranks 6thin the global prevalence of oral health conditions and affects 11% of the world population. It is initiated by the formation of biofilms containing different types of bacteria. These biofilms exist as dental plaque and contain three types of bacteria called Tannerella forsythia, Porphyromonasgingivalis, and Treponema denticola. These three types of bacteria are strongly associated with periodontitis and they are termed as the Red complex. The main focus of the research presented herein is on T. forsythiawhich secrets different types of enzymes that degrade the periodontal tissues. These enzymes are transcribed and translated together from the genes clustered together regulated by an operon system. Periodontal tissues are primarily composed of glycosaminoglycans or (GAGs). These GAGs are linear chains of polysaccharides composed of different disaccharide units. GAGs can exist in conjunction with proteoglycans which are a protein core appended with different types of GAGs The degradative enzymes from T. forsythiadegrades the GAGs connected to the protein core, thus contributing to the destabilization and eventual destruction of the periodontal tissues such as alveolar bone and ligament causing tooth loss. Based on the bioinformatics and enzyme kinetics, it was found that BFO 2291 (chondroitin AC lyase) breaks down chondroitin sulfate A into oligosaccharides containing glucuronic acid and galactosamine. Chondroitin sulfate A is (a type of GAG) one of the main constituents of many periodontal tissues. Enzyme activity was initially analyzed by doing a pH profile using various ranges of buffers and pH values. By doing pH profile, we determined the pH at which this enzyme is the most active which is pH 6.5. Chondroitin AC lyase along with the other enzymes are responsible for the degradation of chondroitin sulfate A using a GAG degradation pathway. The other enzyme analyzed herein was BFO 2294 (KDPG aldolase) which breaks down KDPG (2-keto-3-deoxy-6-phosphogluconate)aldolase into pyruvate and D-glyceraldehyde-3-phosphate. Using a coupled enzymatic assay, it was shown the BFO2294 catalyzed conversion to pyruvate was subsequently converted into lactic acid by lactic dehydrogenase using cofactor NADH which in turn was oxidized to NAD+. In the last step of GAG degradation pathway, KDPG is broken down by KDPG aldolase into pyruvate which can enter into the Krebs cycle and electron transport chain for production of ATP. The final outcome is the production of ATP which is used by the bacteria to drive its cellular functions. Another part of our research was structural characterization of BFO2294 via X-ray diffraction analysis. The BFO2294 enzyme, or KDPG aldolase was successfully crystallized and the diffraction data was collected. KDPG Aldolase catalyzes the reversible cleavage of 2-keto-3deoxy-6-phosphogluconate (KDPG) into pyruvate and D-glyceraldehyde-3-phosphate by retro aldol cleavage. Using various research articles, it was found that the active site of this enzyme contains zwitterionic pair of Glu-49 and Lys-141 which are involved in the catalytic reaction between the enzyme (KDPG aldolase) and the substrate (KDPG)