N-terminal residues are crucial for quaternary structure and active site conformation for the phosphoserine aminotransferase from enteric human parasite E. histolytica

Abstract Phosphoserine aminotransferase (PSAT) is a pyridoxal-5′phosphate (PLP)-dependent enzyme that catalyzes the second reversible step in the phosphoserine biosynthetic pathway producing serine. The crystal structure of E. histolytica PSAT ( Eh PSAT) complexed with PLP was elucidated at 3.0 A resolution and the structures of its mutants, Eh PSAT_Δ45 and EhPSAT_Δ4, at 1.8 and 2.4 A resolution respectively. Deletion of 45 N-terminal residues ( Eh PSAT_Δ45) resulted in an inactive protein, the structure showed a dimeric arrangement drastically different from that of the wild-type protein, with the two monomers translated and rotated by almost 180° with respect to each other; causing a rearrangement of the active site to which PLP was unable to bind. Deletion of first N-terminal 15 ( Eh PSAT_Δ15) and four 11th to 14th residues ( Eh PSAT_Δ4) yielded up to 98% and 90% decrease in the activity respectively. Absence of aldimine linkage between PLP-Lys in the crystal structure of EhPSAT_Δ4 mutant explains for such decrease in activity and describes the importance of these N-terminal residues. Furthermore, a halide-binding site was found in close proximity to the active site. A stretch of six amino acids (146– N N T IY G –151) only conserved in the Entamoeba genus, contributes to halide binding may explain that the halide inhibition could be specific to Entamoeba.