Molecular Basis for the Local Conformational Rearrangement of Human Phosphoserine Phosphatase

Abstract

Human phosphoserine phosphatase (HPSP) regulates the levels of glycine and D-serine, the putative co-agonists for the glycine site of the NMDA receptor in the brain. Here, we describe the first crystal structures of the HPSP in complexes with the competitive inhibitor 2-amino-3-phosphonopropionic acid (AP3) at 2.5 Å, and the phosphate ion (Pi) and the product uncompetitive inhibitor L-serine (HPSP⅐L-Ser⅐Pi) at 2.8 Å. The complex structures reveal that the open-closed environmental change of the active site, generated by local rearrangement of the ␣-helical bundle domain, is important to substrate recognition and hydrolysis. The maximal extent of this structural rearrangement is shown to be about 13 Å at the L4 loop and about 25°at the helix ␣3. Both the structural change and mutagenesis data suggest that Arg-65 and Glu-29 play an important role in the binding of the substrate. Interestingly, the AP3 binding mode turns out to be significantly different from that of the natural substrate, phospho-L-serine, and the HPSP⅐L-Ser⅐Pi structure provides a structural basis for the feedback control mechanism of serine. These analyses allow us to provide a clear model for the mechanism of HPSP and a framework for structure-based drug development. Phosphoserine phosphatase (PSP)1 is an important enzyme in the phosphorylated pathway of serine biosynthesis, which contributes a major portion of the endogenous L-serine (1, 2). In the mammalian nervous system, D-serine is converted from L-serine by serine racemase (3, 4) and acts as a co-agonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, a major neurotransmitter receptor family (5). NMDA receptors require coactivation at a glycine site where D-serine, present in high levels in the mammalian brain, is three times more potent than glycine (6). Recently, a subset of NMDA receptors has been found to be activated by glycine or D-serine in the absence of glutamate (7). The enzymatic reaction of PSP is Mg 2ϩ -dependent and results in the dephosphorylation of phospho-Lserine with the formation of a phosphoenzyme intermediate, which is subsequently autodephosphorylated. The resulting product, L-serine, is not only a precursor for the biosynthesis of glycine but also an uncompetitive inhibitor for the enzymatic reaction of PSP (8). It may be possible to regulate NMDA activity by using selective inhibitors against serine racemase and/or PSP.The PSP family and P-type ATPases are members of the haloacid dehalogenase-like hydrolase family (9 -11). The crystal structures have been elucidated for several members of the haloacid dehalogenase superfamily: the L-2-haloacid dehalogenases, CheY, P-type ATPase and, recently, Methanococcus jannaschii (MJ) PSP (12-16). Three conserved motifs have been observed in amino acid sequences in members of the haloacid dehalogenase superfamily, and these residues are located in the active pocket. The significance of these motifs was confirmed by an assay of mutants for substrate hydrolysis (17). Other phosphoesters, including phosphothreo...

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

“…The previously determined crystal structures of PSP have revealed that PSP belongs to the HAD-like hydrolase fold (30,31). As expected, the overall structure of ThrH is similar to that of PSP.…”

“…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.…”

Mutations responsible for 3-phosphoserine phosphatase deficiency

“…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

“…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).…”