Abstract:
We have investigated the role of cofactor PLP, and its binding domain in stability and subunit assembly of Phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. The presence of cofactor influences the tertiary structure of EhPSAT due to significant differences in the tryptophan microenvironment and proteolytic pattern of apo- and holo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. The stability of enzyme is significantly affected by the presence of cofactor as holo-enzyme shows higher Tm and Cm values for thermal and GdnHCl-induced denaturation respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although, urea dependent unfolding of both apo- and holo-EhPSAT is a three state process, the intermediates stabilized during unfolding are significantly different. For apo-EhPSAT, a monomeric intermediate is stabilized whereas, for holo-EhPSAT a dimeric holo-intermediate is stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild type enzyme. To the best of our knowledge this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.
