Isolation and characterization of the gene encoding d-lactate dehydrogenase enzyme from bacteroides fragilis that is known to be localized in colon cancer tissues

Bacteroides fragilis have been studied for years and more recent studies show association of Bacteroides fragilis with colorectal cancer, which is the third most common cancer worldwide. This bacterium could be grouped under two different strains; nontoxigenic, beneficial to humans, and enterotoxigenic, responsible for the diseases, Bacteroides fragilis. Antibiotic resistance against Bacteroides fragilis infections throughout the world was reported. Therefore, it is essential to work on finding new approaches to combat bacterial infections. In this thesis study, inhibition of enterotoxigenic Bacteroides fragilis metabolism was aimed through targetting D-lactate dehydrogenase enzyme as the bacterium has anaerobic metabolism. L-lactate dehydrogenase was searched in the complete genome of Bacteroides fragilis. Despite the report of presence of L-lactate dehydrogenase in it?s genome, evidences are presented in this study that there was no L-lactate dehydrogenase enzyme in Bacteroides fragilis. Gene sequence of enterotoxigenic Bacteroides fragilis D-lactate dehydrogenase was not determined up to this study. D-lactate dehydrogenase was cloned and expressed as active protein for the first time in literature. Nontoxigenic Bacteroides fragilis D-lactate dehydrogenase was also cloned into pGEM-T Easy vector and both cloned genes were composed of 1011 base pairs including start and stop codons. Both genes were compared for differences; there was an eleven difference at nucleotide level and three at the amino acid level. Bacteroides fragilis D-lactate dehydrogenase gene was subcloned into pLATE31 vector having C-terminal 6 histagged sequence and expressed in Escherichia coli BL21(DE3) cells as a 37.6 kDa protein. The best expression conditions were determined as induction of cells with 0.1 mM IPTG, and growing the cells at 25°C for 5 hours. Affinity chromatography method was used for purifying the enzyme with Ni-NTA agarose column. SDS-PAGE analysis of protein showed presence of high quantity of protein at 19.8 mg/liter with the purity of over 95%. Kinetic characterization was performed at pH: 7.5, 25°C. Grafit 3.0 program was used to determine staedy state kinetic parameters of the purified enzyme. Vmax, Km, kcat, kcat/km, was found to be 0.1594, 0.3752 mM, 32.4 s¯¹, 86.35 mM¯¹s¯¹, respectively, by using pyruvate as substrate. These parameters were also determined for the first time from this enzyme. Homology modelling of Bacteroides fragilis D-lactate dehydrogenase was performed using the same enzyme?s crystal structure from Lactobacillus helveticus as template. This study was followed by molecular docking studies using this proposed model. Mezlocillin, one of the antibiotics used to treat Bacteroides fragilis infections, was found to fit the best into the NADH pocket. All these findings may enable the application of structure based drug design studies towards development of enzyme inhibitors to combat Bacteroides fragilis infections known to be related with colon cancer.