Role of trehalose on structural and functional stability of D-lactate dehydrogenase enzyme

Introduction & Objectives: Lactate dehydrogenase (LDH) belongs to the family of oxidoreductases and catalyzes the reduction of NADH and pyruvate to NAD+ and D-lactate in the final step of anaerobic glycolysis. D-Lactate dehydrogenase (D-LDH) is a marker of common injuries and diseases such as kidney stones, heart failure, some types of cancers and appendicitis. Accordingly, biosensor design and construction is very significant to measure blood lactate levels. The thermal sensitivity of D-LDH is limiting the usage of this protein in certain clinical and diagnostic applications. Trehalose has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation. In this work, trehalose was examined as a support for increasing the thermal stability of D-LDH.Materials & Methods: In this work, the effect of different concentrations of trehalose on the stabilization of recombinant D-LDH was studied. The Specific activity of free D-LDH and D-LDH/trehalose was calculated using the reduction of absorbance in 340 nm due to the oxidation of NADH. The kinetic and thermodynamic parameters, optimum temperature and pH, and the intrinsic fluorescence of free and treated enzymes were examined. Also, an attempt was made to investigate the effect of trehalose on the conformation of UOX in the atomistic level using molecular dynamics (MD) simulations.Results: Additives like trehalose protect the D-LDH conformation, increases its activity and also, improves its stability at high temperatures. Trehalose is a suitable additive for stabilization of the D-LDH. The activity of the enzyme was increased in the presence of trehalose. Also, the RMSD, RMSF and secondary structure analysis indicated that the trehalose protects the enzyme conformation.Conclusion: Results show that trehalose protects the D-LDH conformation and improves enzyme stability at high temperatures. Therefore, trehalose is a suitable additive for stabilization of the D-LDH.

Keywords:

D-Lactate dehydrogenase , stabilization , trehalose , molecular dynamics simulation