The structure of the Mg 2+ -dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the divalent cation in the active site of phosphatases. Most interesting is the biochemical observation that a Ca 2+ ion inhibits the activity of HPSP, even in the presence of added Mg 2+ . Human phosphoserine phosphatase (HPSP) catalyses the last and irreversible step of the de novo biosynthesis of L-serine, i.e. the hydrolysis of phosphoserine leading to the formation of L-serine and inorganic phosphate (Pi). HPSP is a member of the haloacid dehalogenase (HAD) superfamily of which the members are characterized by three short conserved sequence motifs (Fig. 1). The residues of these motifs cluster together to form the active site. All enzymes of the HAD superfamily use the aspartate residue of the first conserved DXXX(T/V) motif as a nucleophilic residue for catalysis [1]. The second motif contains a conserved serine or threonine residue, and the third motif contains a strictly conserved lysine residue followed, at some distance, by less conserved residues and a strictly conserved aspartate. Mutagenesis studies on these conserved residues show that all three motifs play an important role in the catalytic process [2][3][4].Despite the low overall sequence homology among the enzymes of the HAD superfamily, all known structures of enzymes of this superfamily display a conserved fold [5] . Two interesting questions arise from these observations: is there structural evidence for the fact that Mg 2+ in the active site cannot be replaced by another divalent cation without loss of activity, and how does an enzyme manage to select a specific cation from the surrounding fluids that contain a broad variety of cations? A detailed study of the structure of the active site of HPSP with Ca 2+ bound may provide an insight into the biological Correspondence to A. Rabijns,

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.

We report the identification of the mutations in the only known case of L-3-phosphoserine phosphatase deficiency, a recessively inherited condition. The two mutations correspond to the replacement of the semiconserved Asp32 residue by an asparagine and of the extremely conserved Met52 by a threonine. The effects of both mutations were studied on the human recombinant enzyme, expressed in Escherichia coli. Met52Thr almost abolished the enzymatic activity, whereas the Asp32Asn mutation caused a 50% decrease in Vmax. In addition, L-serine, which inhibits the conversion of [ 14 C] phosphoserine to serine when catalysed by the wild-type enzyme, had a lesser inhibitory effect on the Asp32Asn mutant, indicating a reduction in the rate of phosphoenzyme hydrolysis. These modifications in the properties of the enzyme are consistent with the modification in the kinetic properties observed in fibroblasts from the patient.

Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.

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