Isolation, cloning and analysis of the gene encoding lactate dehydrogenase enzyme from Fusobacterium nucleatum

Fusobacterium nucleatum is a gram-negative and obligative anaerobic bacteria species of fusobacteria phylum. This bacterium is invasive, proinflammatory and related with bowel disease, periodontitis and pericarditis. It is also known that F. nucleatum could be associated with colorectal cancer that is responsible for 610,000 deaths per year worldwide, according to the World Health Organisation. F. nucleatum metabolism needs to be further studied with the ultimate aim of blocking bacterial infection. Lactate dehydrogenase enzyme has a key role in anaerobic glycolysis and producing intracellular biomolecules in the bacterium. The enzyme has been chosen as a possible target for new drug design studies. In this thesis study, the gene encoding lactate dehydrogenase from F. nucleatum (FnLDH) has been successfully isolated from the genomic DNA by PCR and then cloned into the pGEM-T easy vector system using the standard protocols for the first time in the literature. FnLDH was analyzed at the nucleotide and amino acid levels. It was determined that the gene was consisted of 957 base pairs. Alignment of amino acid sequence of FnLDH to its counterpart in human showed a distinct sequence difference in the active site of the enzyme that is located between residues 98-109. FnLDH gene was then subcloned into expression vector pKK223-3 and expressed in E. coli JM109 cells. A decent amount of protein was observed in the supernatant fraction, but most of the protein was found to be present in the pellet fraction following the sonication. Production of the active and soluble protein following expression of the enzyme in an appropriate system is essential for the structure based drug design studies. Therefore, in this present study, 34.697 Da protein was observed on SDS PAGE following the protein purification. Because satisfying production of FnLDH protein was not observed by using this system, FnLDH was then cloned into the pLATE 31 vector in order to use advantages of aLICator LIC expression system. Protein was successfully expressed in soluble fraction and purified with over 95% purity using Ni-NTA agarose by affinity chromatography. The purified 34.697 Da protein was analyzed by SDS-PAGE. Specific LDH activity of purified enzyme was measured using NADH and substrate pyruvate at 340 nm. The enzyme was made available for further kinetic characterizations. Homology modeling based on comparative modeling of protein three dimensional structures was used to generate a model of FnLDH using crystal structure of L-lactate dehydrogenase from Lactobacillus casei Identity between two sequences were 41%. Constructed 3D structure was built by MODELLER that models 3D dimensional structures with known structures. ERRAT, ProSA, RAMPAGE and HYPERCHEM softwares were used for the purpose of evaluation and refinement of 3-dimensional structure. Probable ligand binding sites on the protein were determined using web based software, Q-SiteFinder, Pocket-Finder and DoGSiteScorer. The region located between residues 98 and 109 was determined as most favoured ligand binding site. Modelling study further showed the same region being distinct from the same region present in human muscle LDH. Present study may help to understand role of lactate dehydrogenase enzyme in Fusobacterium nucleatum energy metabolism and enables application of further studies on structure based drug design.