Structural and Biochemical Studies of Serine Acetyltransferase Reveal Why the Parasite Entamoeba histolytica Cannot Form a Cysteine Synthase Complex

Abstract

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å , in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica.In bacteria and plants, L-cysteine is synthesized from L-Serine by two key enzymes serine acetyltransferase (SAT) 3 and O-acetyl serine sulfhydrylase (OASS). SAT converts L-Serine to O-acetyl serine by transferring an acetyl group from acetylCoA. Later, OASS converts the O-acetyl serine (OAS) to L-cys-

Three-stage Assembly of the Cysteine Synthase Complex from Escherichia coli

“…Structural work has shown that SAT is a hexamer (a dimer of homotrimers (21,23)) and that OASS is a dimer (24 -26); hence, the SAT: OASS stoichiometry in the complex is 1:2. While this stoichiometry has been called into question (23,(27)(28), the consensus appears to be that the CSC consists of one OASS per SAT trimer (9, 29 -31), a position well supported by the current work.…”

Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from Entamoeba histolytica Assists in Partial Regaining of Feedback Inhibition by Cysteine

“…CS complex formation is absent in E. histolytica, and the feedback inhibition seems to be the only regulatory pathway in the protist pathogen. E. histolytica thus appears to be solely dependent on cysteine for anti-oxidative defense [6], [7], [8].…”

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

“…However, structural and biochemical features of the complex have been elucidated by surface plasmon resonance, steady-state fluorescence, site-directed mutagenesis, two-hybrid system, pre-steady state kinetics, molecular modeling and peptide-binding studies [19–23, 25, 27, 28, 47, 49–61]. Mino and co-workers first identified the C-terminal ten amino acid residues of CysE as critical for CysK-binding [22].…”

“…Inspection of the three-dimensional structure of MNYDI peptide in complex with HiCysK shows that the aromatic P2 side-chain occupies a large hydrophobic pocket that is left partially unoccupied by the side chain of Leu in the wild-type peptide (Figures 3A and 3B). Aromatic residues at position P2 were also reported to increase the affinity of Entamoeba hystolytica CysE (EhCysE) for EhCysK [61]. In E. hystolytica , the CSC is quite unstable and probably does not form under physiological conditions.…”

“…In E. hystolytica , the CSC is quite unstable and probably does not form under physiological conditions. However, Ser→Asp and Pro→Trp mutations at positions P1 and P2 (respectively) significantly increase the affinity of EhCysE for EhCysK [61]. Interestingly, proteobacterial CysE enzymes never carry an aromatic residue at position P2, whereas nearly all CysK moonlighting partners have Tyr, Phe or His residues at this position.…”