Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship

Abstract

Background: Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme.

Findings: EhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed.

Conclusions: The study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.

Figure 1

(A) Alignment of the amino acid sequence of PSAT from Bacillus circulans spp. Alkalophilus (BCIR), Bacillus alcalophilus (BALC), Entamoeba histolytica (EHIS), and E coli (ECOL). Highlighted letters indicate active site residues that interact with the cofactor PLP (light grey) and Cl^- ions (dark grey). The substitution of Pro201 (BCIR PSAT) to Ala195 (EhPSAT) in the loop region has been highlighted in blue. (B) Homology model of EhPSAT dimer visualized through UCSF chimera. The cofactor PLP present in the active site is represented in cyan color. The inset shows stacking of the cofactor PLP(cyan color) and the Trp101(brown color) at the active site.

Figure 2

Overexpression, purification, functional activity and pH dependent structural changes of EhPSAT. (A) SDS-PAGE analysis of E coli lysate over expressing EhPSAT and the purified protein. Lanes 1-4 represent molecular weight markers, uninduced culture, induced culture and purified protein, respectively. (B) pH-dependent enzymatic activity profile of EhPSAT. The data has been represented as percentage relative activity with highest activity observed at pH 8.5 taken as 100%, each point representing mean ± SD of three independent measurements. (C) pH induced changes in the secondary structure of EhPSAT. The effect of pH on the CD signal at 222 nm. The inset shows far-UV CD spectra at pH 6 (solid line), 7 (dashed line), 8 (dotted line) and 9 (dash-dotted line), respectively. (D) and (E) pH-induced changes in PyP and Trp fluorescence polarization, respectively. In both the panels the filled and the open symbols represent protein samples in absence and presence of 200 mM NaCl, respectively. (F) pH induced changes in fluorescence emission spectra of EhPSAT excited at 295 nm. The curves 1-4 represent protein samples incubated at pH 6, 7, 8 and 9, respectively.

Figure 3

pH induced compaction of EhPSAT, oligomeric state and thermal stability. (A) Curves 1-4 represent SEC profile of EhPSAT on a superdex 200, 10/300 GL column at pH 6, 7, 8 and 9, respectively. The inset shows the graph of elution volume plotted against standard molecular mass markers. The proteins are (1) 440 KDa (ferritin), (2)158 KDa (aldolase), (3) 75 KDa (conalbumin) and (4) 43 KDa (ovalbumin). (B) 10% SDS-PAGE profile of glutaraldehyde crosslinked sample of EhPSAT incubated at different pH. Lane 1-6 represent molecular weight markers, uncrosslinked EhPSAT, and glutaraldehyde crosslinked form of EhPSAT at pH 9, 8, 7 and 6, respectively. Cooperative thermal unfolding of EhPSAT at pH 8.5 (panel C) and 6 (panel D) as measured by loss of CD ellepticity at 222 nm and 415 nm. A linear extrapolation of baselines in pre and post transition regions was used to determine the fraction unfolded protein within the transition region by assuming a two state mechanism of unfolding. The open and filled circle represent for far and near UV-CD signals, respectively. The thermal transition of the enzyme was found to be irreversible with precipitation observed at the end of the scan.

Figure 4

Effect of Sodium halides on pH dependent changes in functional activity and structural features of EhPSAT. (A) Inhibition of functional activity in presence of 200 mM concentration of various Sodium halides. The activity of EhPSAT in absence of salt (native protein) at pH 8.5 was taken as 100%. (B) pH dependent enzymatic activity profile of EhPSAT in presence of 200 mM concentration of NaCl (rectangles) and NaF (circles), respectively. For samples in presence of NaCl and NaF the highest value observed was taken as 100%. Fluorescence emission spectra of EhPSAT in presence of 200 mM NaCl (Panel C) and NaF (Panel D) at different pH. The different curves in both the panel represent protein samples at pH 6 (solid line), 7 (dashed line), 8 (dotted line) and 9 (dash dotted line). SEC profile at various pH in presence of NaCl (panel E) and NaF (panel F). The curves 1-4 represent SEC profile in presence of 200 mM concentration of salts at pH 6, 7 and 8, and 9 respectively.

Figure 5

Thermal unfolding in presence of Sodium halides. Thermal denaturation profile at pH 8.5 in presence of 200 mM NaCl (panel A), NaF (panel B) and at pH 6 in presence of 200 mM NaCl (panel C) and NaF (panel D), respectively. The open and closed circles represent CD ellepticity measured at 222 nm and 415 nm, respectively. A linear extrapolation of baselines in pre and post transition regions was used to determine the fraction unfolded protein within the transition region by assuming a two state mechanism of unfolding. The thermal transition of the enzyme was found to be irreversible with precipitation observed at the end of the scan.