Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Characterization of the phosphoserine of pepsinogen using 31P nuclear magnetic resonance: corroboration of X-ray crystallographic results

The endogenous phosphoserine residue in porcine pepsinogen has been titrated with use of phosphorus-31 nuclear magnetic resonance (31P NMR). It has an observed pKa2 of 6.7 and a narrow line width (congruent to 10 Hz). The phosphate can be readily removed by an acid phosphatase from potato; however, it is resistant to hydrolysis by several alkaline phosphatases. The X-ray crystal structure of porcine pepsinogen at 1.8-A resolution [James, M. N. G., & Sielecki, A. (1986) Nature (London) 319, 33-38] shows a rather weak and diffuse region of electron density in the vicinity of the phosphorylated serine residue. This suggests considerable dynamic mobility or conformational disorder of the phosphate. In order to define more fully this behavior, the NMR data have been used to corroborate these crystallographic results. All these physical data are consistent with a highly mobile phosphoserine residue on the surface of the zymogen and freely exposed to solvent. In addition, certain properties of this phosphoserine moiety on pepsinogen are similar to those of one of the phosphorylated residues of ovalbumin. The possible significance of this is discussed.     

Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications

We report here the crystal structure of a trehalose-6-phosphate phosphatase-related protein (T6PP) from Thermoplasma acidophilum, TA1209, determined by the dual-wavelength anomalous diffraction (DAD) method. T6PP is a member of the haloacid dehalogenase (HAD) superfamily with significant sequence homology with trehalose-6-phosphate phosphatase, phosphoserine phosphatase, P-type ATPases and other members of the family. T6PP possesses a core domain of known alpha/beta-hydrolase fold, characteristic of the HAD family, and a cap domain, with a tertiary fold consisting of a four-stranded beta-sheet with two alpha-helices on one side of the sheet. An active-site magnesium ion and a glycerol molecule bound at the interface between the two domains provide insight into the mode of substrate binding by T6PP. A trehalose-6-phosphate molecule modeled into a cage formed by the two domains makes favorable interactions with the protein molecule. We have confirmed that T6PP is a trehalose phosphatase from amino acid sequence, three-dimensional structure, and biochemical assays.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

Tk-PTP,protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.     

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation,membrane association,and enzyme function in yeast

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.     

Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser-56

The site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp-57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser-->Ala substitution at this position in wild-type CheY had no effect on function. However, replacement of Ser-56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser-56 or Asp-57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid-base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu-57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.     

The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99-102]. In this study, we report that the heterologously expressed Mj0968-His(10) protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not affect the V(max), but only the K(M) of the reaction. Labeling studies with [gamma-(32)P]ATP and [alpha-(32)P]ATP revealed that the previously reported labeling experiments [Ogawa et al., 2000] do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K(d) values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme.     

Ca2+ Induces Spontaneous Dephosphorylation of a Novel P5A-type ATPase

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mm) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.     

Crystal structures of the substrate free-enzyme, and reaction intermediate of the HAD superfamily member, haloacid dehalogenase DehIVa from Burkholderia cepacia MBA4

DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.     

Sites phosphorylated in bovine cardiac troponin T and I. Characterization by 31P-NMR spectroscopy and phosphorylation by protein kinases

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P-NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L.M.G. Jr (1988) Eur. J. Biochem. 176, 327-334]. Troponin I, which contains phosphate covalently linked to serine-23 and/or -24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335-342], shows three resonances. Mg2(+)-saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP- and cGMP-dependent protein kinases as well as for protein kinase C. Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N-terminus. An additional phosphoserine is located in the C-terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine-194, thus demonstrating exposure of this residue on the surface of holotoponin.     

Analysis of the in vivo phosphorylation state of protein phosphatase inhibitor-2 from rabbit skeletal muscle by fast-atom bombardment mass spectrometry

A new procedure has been developed for identifying phosphoserine residues in proteins, and is used to analyse the in vivo phosphorylation state of inhibitor-2. The method employs reverse-phase liquid chromatography to resolve phosphorylated and dephosphorylated forms of peptides and fast-atom bombardment mass spectrometry (FABMS) to identify phosphorylated derivatives. The positions of phosphorylation sites within peptides are located by gas-phase sequencer analysis after conversion of phosphoserine residues to S-ethylcysteine. The phosphorylation sites on inhibitor-2 were identified as serines-86, -120 and -121, the three residues phosphorylated in vitro by casein kinase-II. Serine-86 was phosphorylated to 0.7 mol/mol and serines-120 and -121 each to 0.3 mol/mol. These values were not altered significantly by intravenous injection of adrenalin or insulin. No phosphate was present in the region comprising residues 1-49, even after injection of adrenalin, demonstrating that inhibitor-2 is not a substrate for cyclic AMP-dependent protein kinase in vivo. The absence of phosphotyrosine also indicated that inhibitor-2 is not a physiological substrate for the insulin receptor. Surprisingly, no phosphate was present at threonine-72, the residue phosphorylated in vitro by glycogen synthase kinase-3, after injection of either propranolol, adrenalin or insulin. The implications of this finding for the in vivo activation of protein phosphatase 1I (the 1:1 complex between inhibitor-2 and the catalytic subunit of protein phosphatase-1) are discussed. FABMS analysis of inhibitor-2 confirmed the accuracy of the primary structure reported previously, and showed that the only post-translational modifications were an N-acetyl moiety and the three phosphoserine residues. FABMS also demonstrated the presence of an additional serine residue at the C-terminus, and showed that 50% of isolated inhibitor-2 molecules lack the C-terminal Ser-Ser dipeptide.     

Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as ³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.     

The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.     

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.     

Characterization of the dimerization domain in the FNR transcription factor

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.     

Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase

The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.     

Tyr-179 and Lys-183 are essential for enzymatic activity of 11 beta-hydroxysteroid dehydrogenase.

Tyr-179 and Lys-183 are likely to be functionally important residues in 11 beta-hydroxysteroid dehydrogenase, as these amino acids are absolutely conserved in all members of the "short chain dehydrogenase" family. We modified these residues by site-directed mutagenesis of rat cDNA and transfected these constructs into CHO cells. A highly but not absolutely conserved residue, Asp-110, was also studied. Mutation of Tyr-179 to Phe or Ser completely abolished enzymatic activity (interconversion of corticosterone and 11-dehydrocorticosterone), as did Lys-183-->Arg. Asp-110-->Asn affected activity only mildly. Tyr-179 and Lys-183 may be directly involved in the catalytic function of this class of enzymes.     

Insights into the catalytic mechanism of human sEH phosphatase by site-directed mutagenesis and LC-MS/MS analysis

We have recently reported that human soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a structural relationship with other members of the haloacid dehalogenase superfamily, the sEH N-terminal phosphatase domain revealed four conserved sequence motifs, including the proposed catalytic nucleophile D9, and several other residues potentially implicated in substrate turnover and/or Mg²⁺ binding. To enlighten the catalytic mechanism of dephosphorylation, we constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A total of 18 mutants were constructed and recombinantly expressed in Escherichia coli as soluble proteins, purified to homogeneity and subsequently analysed for their kinetic parameters. A replacement of residues D9, K160, D184 or N189 resulted in a complete loss of phosphatase activity, consistent with an essential function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 leads to sEH mutant proteins with altered kinetic properties. We further provide evidence of the formation of an acylphosphate intermediate on D9 by liquid chromatography-tandem mass spectrometry based on the detection of homoserine after NaBH₄ reduction of the phosphorylated enzyme, which identifies D9 as the catalytic nucleophile. Surprisingly, we could only show such homoserine formation using the D11N mutant, which strongly suggests D11 to be involved in the acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes rate limiting, which then allows trapping of the labile intermediate. Substrate turnover in the presence of ¹⁸H₂O revealed that the nucleophilic attack during the second reaction step occurs at the acylphosphate phosphorous. Based on these findings, we propose a two-step catalytic mechanism of dephosphorylation that involves the phosphate substrate hydrolysis by nucleophilic attack by the catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme intermediate supported by D11.     

Plant P-type ATPases

P-type ATPases are a superfamily of membrane proteins involved in many physiological processes that are fundamental for all living organisms. Using ATP, they can transport a variety of ions and other substances across all types of cell membranes against a concentration electrochemical gradient. P-type ATPases form a phosphorylated intermediate and are sensitive to vanadate. Based on evolutionary relations and sequence homology, P-type ATPases are divided into five major families. All P-type ATPases share a simple structure and mechanism, but also possess domains characteristic for each family, which are crucial for substrate specificity. These proteins usually have a single subunit with eight to twelve transmembrane segments, a large central cytoplasmic domain with the conservative ATP binding site along with N and C termini exposed to the cytoplasm. Because of variety of proteins that belong to P-type ATPase superfamily, in this review the comparison of functional and structure properties of plant cells P-type ATPases is presented, as well as their important role in adaptation to environmental stress.