Cloning and analysis of the gene encoding enolase enzyme from fusobacterium nucleatum

Fusobacterium nucleatum is related to periodontal diseases such as gingivitis and periodontitis in human. It is also one of the prevalent species in the oral microbiome and interacts with other bacteria in oral flora and host epithelial cells. Antibiotic susceptibility in Fusobacteria has been reported, so this showed that development of alternative drugs against this species is required. Enolase is an enzyme which is responsible from a reaction in glycolytic pathway of F. nucleatum and converts 2-phosphoglyceric acid (2-PGA) to phosphoenolpyruvate (PEP). In this study, F. nucleatum enolase was chosen as a target molecule because of its role in the metabolism of the bacteria. The enzyme was isolated, cloned, expressed and analysed both kinetically and structuraly, for the first time in the literature. The gene encoding enolase from F. nucleatum (FnENO) was first cloned into pGEM®-T Easy Vector system and then sub-cloned into pLATE 31 vector for the expression of enzyme in Escherichia coli BL21 (DE3) cells. 6xHis-tag region was added to the C-terminal of the protein to enable purification of the protein by affinity chromotography using Ni-NTA agarose. SDS-PAGE analysis of the 46,37 kDa protein showed that the enzyme was obtained at high purity, over >% 95, following the protein purification process. Optimum pH and thermostability analyses were performed prior to kinetic characterization of the enzyme. It was found that the optimum pH was 8,5 for this enzyme and specific activity was calculated as 57,14 U/mg. Thermostability analyses showed that FnENO activity remained stable between 30-60 0C. Kinetic analysis was performed at pH 8,5 and 250C. Staedy state kinetic parameters of purified FnENO was determined using GraFit 3.0. Kinetic values were obtained using 2-PGA as substrate; Km: 0,4825 mM, Vmax: 0,1587, kcat: 20,38 s-1 and kcat/Km: 4,22 x 104 M-1s-1. Homology modelling of Fusobacterium nucleatum enolase enzyme was built using Enterococcus hirae enolase structure as template and possible drug target regions were described. All these results may open new insights towards structure based drug design studies and enolase may be a candidate molecule to combat Fusobacterium nucleatum infections in human. Keywords: Fusobacterium nucleatum, enolase, periodontal diseases, kinetic characterization, structure based drug design.