Unraveling the Structural Basis of Urea-induced Unfolding of Fasciola gigantica Cytosolic Malate Dehydrogenase

Abstract Malate dehydrogenase enzyme plays an essential role in energy metabolism in all eukaryotes. It catalyzes the interconversion of malate to oxaloacetate, producing NAD+/NADH. In the current study, we monitored the urea-induced conformational changes in Fasciola gigantica malate dehydrogenase (FgMDH) structure using multiple spectroscopic techniques combined with all-atom molecular dynamics (MD) simulations. FgMDH showed a two-state cooperative unfolding revealed by intrinsic tryptophan (Trp) fluorescence and CD spectroscopy; however, their non-superimposable curves indicate a partially folded intermediate state formation. The study showed that the unfolding of tertiary structures preceded the secondary structure unfolding. The binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) at 1.5 M urea confirmed stabilization of the intermediate state. The free energy of stabilization determined from Trp fluorescence and CD spectroscopy were 11.4 and 13.2 kcal.mol-1, respectively. The loss of enzymatic activity was observed before any significant loss in structural components. This suggested that low urea concentrations induce minor structural changes in the substrate or cofactor binding site leading to activity loss. Quenching of fluorescence intensity also provides information on unfolding and activity loss. MD simulations confirmed that FgMDH formed an intermediate state at 1.5 M urea at 300 K, which destabilized at higher urea concentration and temperature due to disrupted hydrogen-bond networks and hydrophobic interactions. We conclude that a partially folded metastable state is stabilized during the unfolding of cytosolic FgMDH. A strong correlation was observed between the data obtained from in-vitro and in-silico approaches.