High-resolution structure of human phosphoserine phosphatase in open conformation

Abstract

The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53 A. The crystals are orthorhombic, belonging to space group C222(1), with unit-cell parameters a = 49.03, b = 130.25, c = 157.29 A. The asymmetric unit contains two molecules. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (R(free) = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the l-serine biosynthesis pathway. It catalyses the Mg2+-dependent hydrolysis of l-phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion.

Mutations responsible for 3-phosphoserine phosphatase deficiency

“…The presence of a methionine in this position could indeed stabilize the disordered region and promote refolding to form helix 3 (helix 2 in the M. jannaschii enzyme), which closes the catalytic site. 18 This role, and the high conservation of this residue, is consistent with the dramatic effect of the Met52Thr mutation, which almost abolishes the enzymatic activity.…”

“…9 Interpretation of the effect of the two mutations Structure analysis of Methanococcus jannaschii phosphoserine phosphatase indicates that Met43, which aligns with Met52 in the human enzyme, contacts the substrate in a closed conformation of the protein. 16 In the human structures, which are in slightly different open conformations, 17,18 Met52 and the surrounding amino acids in the sequence are disordered, presumably allowing phosphoserine to access the catalytic site and the products to leave it. It has been proposed that the extremely conserved methionine is an important factor in switching the enzyme from the open to the closed conformation by interacting with the substrate.…”

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

“…However, inclusion of Zn 2ϩ ion in the assay buffer abolished SerB2 enzymatic activity in a dose-dependent manner that might be attributed to disruption of the SerB2 secondary structure, thereby preventing binding of O-phospho-L-serine to its active site. We also observed reduction in SerB2 activity upon inclusion of Ca 2ϩ ion in the assay buffer, which might be attributed to disruption of nucleophilic attack by the conserved Asp residue (motif I) on the phosphate group of O-phospho-L-serine as reported earlier in the case of HPSP (22). The activity of SerB2 enzyme was enhanced by 15-20% by inclusion of nonionic detergent in assay conditions that might be attributed to stabilization of SerB2 secondary structure.…”

“…These enzymes have an absolute requirement for divalent metal ion, and the conserved Asp residue in motif I forms a phosphoenzyme intermediate with the substrate via nucleophilic attack (19,20). HAD family of enzymes possess the Rossmannoid ␣/␤ fold and an inserted "cap" that regulates substrate access to its active site (21)(22)(23). PSP enzymes have been extensively biochemically characterized in various microorganisms and have been demonstrated to facilitate entry of Porphyromonas gingivalis into host cells by modulating host cytoskeletal architecture, innate immune responses, and dephosphorylating colicin and NF-〉 (24 -26).…”

The thrH Gene Product of Pseudomonas aeruginosa Is a Dual Activity Enzyme with a Novel Phosphoserine:Homoserine Phosphotransferase Activity

“…Substrate binding-induced conformational changes that results in a "closed" active site have been a common theme in several HAD superfamily enzymes (9,31,36). The conformational differences between the two independent ThrH monomers in the asymmetric unit demonstrated the structural flexibility of ThrH especially at the hinge region between domains I and II.…”