Serine acetyl transferase (SAT) is one of the crucial enzymes in the cysteine biosynthetic pathway and an essential enzyme for the survival of Entamoeba histolytica, the causative agent of amoebiasis. E. histolytica expresses three isoforms of SAT, where SAT1 and SAT2 are inhibited by the final product cysteine, while SAT3 is not inhibited. SAT3 has a slightly elongated C-terminus compared to SAT1. To understand the stability and conformational transition between two secondary structures of proteins, we measured the effect of urea, a chemical denaturant, on two isoforms of SAT (SAT1 and SAT3) of E. histolytica. The effect of urea on the structure and stability of SAT1 and SAT3 was determined by measuring changes in their far-UV circular dichroism (CD), Trp fluorescence, and near-UV absorption spectra. The urea-induced normal transition curves suggested that the structural transition is reversible and follows a two-state process. Analysis of the urea-induced transition of all optical properties for the stability parameters ΔG D° (Gibbs free energy change (ΔG D) in the absence of urea), m (dependence of ΔG D on urea concentration), and C m (midpoint of urea transition) suggested that SAT1 is more stable than SAT3. Characterization of the end product of the urea-induced transition of both proteins by the far-UV CD and Trp-fluorescence and near-UV absorbance suggested that urea causes α-helix to β-sheet transition and burial of Trp residues, respectively. To support the in vitro findings, 100 ns molecular dynamics simulations (in silico study) were performed. Both the spectroscopic and molecular dynamics approaches clearly indicated that SAT1 is more stable than SAT3. SAT3 has evolved to escape the feedback inhibition to keep producing cysteine, but in the process, it compromises its structural stability relative to SAT1.
© 2022 The Authors. Published by American Chemical Society.