: Serine is ubiquitously synthesized in all living organisms from the glycolysis intermediate 3-phosphoglycerate (PGA) by phosphoserine biosynthetic pathway, consisting of three different enzymes, namely: 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Any functional defect or mutation in these enzymes may cause deliberating conditions, such as colon cancer progression and chemoresistance in humans. Phosphoserine aminotransferase (PSAT) is the second enzyme in this pathway that converts phosphohydroxypyruvate (PHP) to O-phospho-L-serine (OPLS). Humans encode two isoforms of this enzyme: PSAT1 and PSAT2. PSAT1 exists as a functional dimer, where each protomer has a large and a small domain; each large domain contains a Lys residue that covalently binds PLP. The PLP-binding site of human PSAT1 and most of its active site residues are highly conserved in all known PSAT structures except for Cys-80. Interestingly, Two PSAT structures from different organisms show halide binding near their active site. While the human PSAT1 shows a water molecule at this site with different interacting residues, suggesting the inability of halide binding in the human enzyme. Analysis of the human PSAT1 structure showed a big patch of positive charge around the active site, in contrast to the bacterial PSATs. Compared to human PSAT1, the PSAT2 isoform lacks 46 residues at its C-terminal tail. This tail region is present at the opening of the active site as observed in the other PSAT structures. Further structural work on human PSAT2 may reveal the functional importance of these 46 residues.

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Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a ~500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+.