Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with K _i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.     

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.     

Analyzing the catalytic mechanism of MPtpA: a low molecular weight protein tyrosine phosphatase from Mycobacterium tuberculosis through site-directed mutagenesis

Mycobacterium tuberculosis adopts various measures to escape from the hostile environment of the host cells. A low molecular weight protein tyrosine phosphatase (LMWPTPase) MPtpA was found to be active in virulent mycobacterial forms during the phagocytosis process. To ascertain the importance of conserved residues Cys11, Arg17, and Asp126 in the catalytic mechanism of MPtpA, site-directed mutagenesis was performed, namely C11S, R17A, D126A, and D126N. Kinetic characterization of wild-type and the mutant MPtpAs using para-nitrophenyl phosphate revealed the reaction mechanism followed by this LMWPTPase and it is similar to the other PTPases. All the LMWPTPases have a common signature motif, 'C(X)(5)R(S/T)' and an Asp as the general acid residue and the mechanism followed by MPtpA can be aptly attributed to other LMWPTPases as well, considering the similar three-dimensional conformation. We have shown that the mutations caused major changes in the chemical environment surrounding the mutated residues and resulted in the decrease of catalytic activity significantly. Inhibition kinetics was performed with phosphate analogues: sodium molybdate, sodium orthovanadate, and sodium tungstate.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Structural basis for selective inhibition of Mycobacterium tuberculosis protein tyrosine phosphatase PtpB

Tyrosine kinases and phosphatases establish the crucial balance of tyrosine phosphorylation in cellular signaling, but creating specific inhibitors of protein Tyr phosphatases (PTPs) remains a challenge. Here, we report the development of a potent, selective inhibitor of Mycobacterium tuberculosis PtpB, a bacterial PTP that is secreted into host cells where it disrupts unidentified signaling pathways. The inhibitor, (oxalylamino-methylene)-thiophene sulfonamide (OMTS), showed an IC(50) of 440 +/- 50 nM and >60-fold specificity for PtpB over six human PTPs. The 2 A resolution crystal structure of PtpB in complex with OMTS revealed a large rearrangement of the enzyme, with some residues shifting >27 A relative to the PtpB:PO(4) complex. Extensive contacts with the catalytic loop provide a potential basis for inhibitor selectivity. Two OMTS molecules bound adjacent to each other, raising the possibility of a second substrate phosphotyrosine binding site in PtpB. The PtpB:OMTS structure provides an unanticipated framework to guide inhibitor improvement.     

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.     

Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase

The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (Wzc_CD). Wzc_CD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from Wzc_CD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine.     

Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects

PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree of phenotypic variation with viability of mutant adult flies ranging from 0 to 96%, and ISNb motor nerve defects ranging from 11 to 57% [Desai and Purdy, 2003]. To determine whether mutations in Ptp69D affecting axon guidance and viability demonstrate losses of phosphatase activity and whether differences in catalytic potential underlie phenotypic variability, we expressed full-length wild-type and mutant PTP69D protein in Schneider 2 cells, and assessed phosphatase activity using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP). Detailed biochemical characterization of wild-type PTP69D, including an examination of sensitivity to various inhibitors, in vitro catalytic efficiency, and the pH-k(cat) profile of the enzyme, suggests a common tyrosine phosphatase reaction mechanism despite lack of sequence conservation in the WPD loop. Analysis of mutant proteins revealed that every mutant had less than 1% activity relative to the wild-type enzyme, and these rates did not differ significantly from one another. These results indicate that mutations in Ptp69D resulting in axon guidance defects and lethality significantly compromise catalytic activity, yet the range of biological activity exhibited by Ptp69D mutants cannot be explained by differences in catalytic activity, as gauged by their ability to hydrolyze the substrate DiFMUP.     

Involvement of the membrane distal catalytic domain in pervanadate-induced tyrosine phosphorylation of receptor protein-tyrosine phosphatase alpha

Receptor protein-tyrosine phosphatase alpha, RPTPalpha, is a typical transmembrane protein-tyrosine phosphatase (PTP) with two cytoplasmic catalytic domains. RPTPalpha became strongly phosphorylated on tyrosine upon treatment of cells with the PTP inhibitor pervanadate. Surprisingly, mutation of the catalytic site Cys in the membrane distal PTP domain (D2), but not of the membrane proximal PTP domain (D1) that harbors the majority of the PTP activity, almost completely abolished pervanadate-induced tyrosine phosphorylation. Pervanadate-induced RPTPalpha tyrosine phosphorylation was not restricted to Tyr789, a known phosphorylation site. Cotransfection of wild-type RPTPalpha did not potentiate tyrosine phosphorylation of inactive RPTPalpha-C433SC723S, suggesting that RPTPalpha-mediated activation of kinase(s) does not underlie the observed effects. Mapping experiments indicated that pervanadate-induced tyrosine phosphorylation sites localized predominantly, but not exclusively, to the C-terminus. Our results demonstrate that RPTPalpha-D2 played a role in pervanadate-induced tyrosine phosphorylation of RPTPalpha, which may suggest that RPTPalpha-D2 is involved in protein-protein interactions.     

'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

Catalytic mechanism of inulinase from Arthrobacter sp. S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k_cat/K_m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k_cat, but not due to variations in K_m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK_a of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK_a. Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.     

Mycobacterium tuberculosis protein tyrosine phosphatase PtpB structure reveals a diverged fold and a buried active site

Intracellular pathogenic bacteria manipulate host signal transduction pathways to facilitate infection. Mycobacterium tuberculosis protein tyrosine phosphatases (PTPs) PtpA and PtpB are thought to be secreted into host cells and interfere with unidentified signals. To illuminate the mechanisms of regulation and substrate recognition, we determined the 1.7 A resolution crystal structure of PtpB in complex with the product phosphate. The protein adopts a simplified PTP fold, which combines features of the conventional PTPs and dual-specificity phosphatases. PtpB shows two unusual elaborations--a disordered, acidic loop and a flexible, two-helix lid that covers the active site--that are specific to mycobacterial orthologs. Biochemical studies suggest that substrate mimicry in the lid may protect the phosphatase from oxidative inactivation. The insertion and deletion of large structural elements in PtpB suggest that, outside the active site module, the PTP family is under unusual selective pressure that promotes changes in overall structure.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

Germline polymorphisms of RET and GFRA1 genes in patients with medullary thyroid carcinoma

The association of RET and GFRA1 polymorphisms with a predisposition to sporadic medullary thyroid cancer (MTC) and their effects on the clinical features of hereditary and sporadic MTC were studied in 67 MTC patients (22 hereditary and 45 sporadic), 3 asymptomatic carriers of mutant RET, and 178 healthy control residents of Russia. RET exons 8, 10, 11, 13, 14, 15, and 16 and intron 1 along with the GFRA1 5′-UTR were screened by PCR and subsequent direct sequencing or RFLP analysis. Eight polymorphic variants of RET (exons 11, 13, 14, and 15 and introns 1, 8, 13, and 14) and four GFRA1 polymorphisms were detected. Linkage disequilibrium was found between RET variants G691S and S904S, L769L and IVS8, and S836S and IVS13. In sporadic MTC the allele frequency of only one polymorphic RET variant, L769L, was significantly lower than in the control group. In hereditary MTC a significant overrepresentation of the S836S and underrepresentation of the S904S polymorphic variants were observed as compared to groups with sporadic MTC and the controls. Cosegregation was not found between individual polymorphisms and the phenotype of sporadic MTC. In patients with hereditary MTC whose genotype had the polymorphic L769L and the wild-type S836S variants, the disease manifested 20 years later, on average, than in individuals with polymorphic L769L and S836S or with wild-type L769L (P = 0.01). The results suggest a protective role of the L769L polymorphism in sporadic MTC and a modulating effect of the combination polymorphic L769L with wild-type S836S on the clinical outcome of hereditary MTC.     

Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling

Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.     

Coupling between the voltage-sensing and phosphatase domains of Ci-VSP

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.     

Topology of Streptococcus pneumoniae CpsC,a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein in S. pneumoniae strain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein''s topology was not as predicted. Similar results were seen with membranes from S. pneumoniae strain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.     

The insertion of (LK) residues at the N-terminus of Staphylococcus xylosus lipase affects its catalytic properties and its enantioselectivity

Two residues (LK) were inserted at the N-terminus of Staphylococcus xylosus lipase (SXL). The S. xylosus lipase wild-type (WT) and its constructed mutant (LK-SXL) were expressed in Escherichia coli BL21 (DE3) and purified to homogeneity using Sephacryl S-200 gel filtration, anion exchange and cation exchange chromatography.A comparative study on the kinetics and interfacial properties of the SXL wild-type and its mutant was performed using emulsified and monolayer substrate. The first important observation was that the insertion of two residues (LK) resulted in an improvement in temperature activity, a better stability, a 3-fold specific activity and a 6-fold catalytic efficiency (k_cat/K_mapp), compared to the SXL wild-type.Using the monolayer technique, this study showed that the inserted residues (LK) at the N-terminus do not affect the adsorption step of the S. xylosus lipase into a monomolecular film of diC₁₂-phosphatidyl-choline.A kinetics study on the surface pressure dependency, stereoselectivity, and regioselectivity of the SXL wild-type and its mutant was also performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Furthermore, the insertion of two residues (LK) at the N-terminus of S. xylosus lipase was shown to be accompanied by the switch of its enantioselectivity.To confirm that the insertion of LK residues at the N-terminus of SXL affects both the hydrolysis properties and the synthesis, new experiments were performed and the production of butyl acetate by SXL before and after LK insertion was assessed. The insertion of these residues may have some valuable applications in industry.     

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.     

A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurlum

A number of bacterial pathogens have evolved sophisticated strategies to subvert host-cell signal-transduction pathways for their own benefit. These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells. In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence. Sequence analysis revealed that a carboxy-terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases. Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine. An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases. In addition, an amino-terminal region of SptP exhibited sequence similarity to the ribosyltransferase exo-enzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp. The modular nature of this effector protein may allow multiple interactions with host-cell signalling functions.