Mn²⁺ Is a Native Metal Ion Activator for Bacteriophage λ Protein Phosphatase^†

Bacteriophage λ protein phosphatase (λPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5‘-nucleotidase, and DNA repair enzymes such as Mre11. λPP can be activated several-fold by various divalent metal ions, with Mn2+ and Ni2+ providing the most significant activation. Despite the extensive characterization of purified λPP in vitro, little is known about the identity and stoichiometry of metal ions used by λPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of λPP. Escherichia coli cells overexpressing λPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express λPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing λPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing λPP exhibit resonances previously attributed to mononuclear Mn2+ and dinuclear [(Mn2+)2] species bound to λPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing λPP indicates the presence of approximately 40 μM mononuclear Mn2+-λPP and 60 μM [(Mn2+)2]-λPP. The data suggest that overexpression of λPP results in a mixture of apo-, mononuclear-Mn2+, and dinuclear-[(Mn2+)2] metalloisoforms and that Mn2+ is a physiologically relevant activating metal ion in E. coli.</i

Inhibition of Bacteriophage λ Protein Phosphatase by Organic and Oxoanion Inhibitors^†

Bacteriophage λ protein phosphatase (λPP) with Mn2+ as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of λPP, albeit with inhibition constants (Ki) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (Ki = 5.1 ± 1.6 μM) whereas phosphonoacetic acid (Ki = 380 ± 45 μM) and acetohydroxamic acid (Ki > 75 mM) modestly inhibited λPP. Low-temperature EPR spectra of Mn2+-reconstituted λPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn2+ ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn2+)2 dimer is most prominent upon the addition of orthovanadate (Ki = 0.70 ± 0.20 μM) and PhAH, indicating that these inhibitors tightly interact with the (Mn2+)2 form of λPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by λPP and other serine/threonine protein phosphatases

Interaction of Bacteriophage λ Protein Phosphatase with Mn(II): Evidence for the Formation of a [Mn(II)]₂ Cluster^†

The interaction of bacteriophage λ protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage λ protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases

Evidence for Differential Binding of Isoniazid by <i>Mycobacterium</i> <i>tuberculosis</i> KatG and the Isoniazid-Resistant Mutant KatG(S315T)

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase−peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position ≈12 Å from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a “type I” substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site

The Transition State of the Phosphoryl-Transfer Reaction Catalyzed by the Lambda Ser/Thr Protein Phosphatase

The catalytic reaction of the Mn2+ form of the native bacteriophage λ phosphatase and the H76N mutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-dependent rate studies. The kinetic isotope effects in the substrate were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom undergoing bond cleavage [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects with native enzyme at the pH optimum of 7.8 were 1.0133 ± 0.0006 for 18(V/K)bridge, 1.0006 ± 0.0003 for 15(V/K), and 0.9976 ± 0.0003 for 18(V/K)nonbridge. These values were constant within experimental error across the pH range from 6.0 to 9.0 and were also unchanged for the slower catalytic reaction resulting when Ca2+ was substituted for Mn2+. The results indicate that the chemical step of P−O bond cleavage is rate-limiting, the first metallophosphatase for which this has been shown to be the case. The isotope effects are very similar to those measured for reactions of protein-tyrosine phosphatases, indicating that the two families of enzymes share similar dissociative transition states. The 18(V/K)bridge and 15(V/K) isotope effects for the H76N mutant were slightly increased in magnitude relative to the native enzyme but were much smaller than the values expected if the leaving group were departing with a full negative charge. The pH vs kcat profile for the native enzyme is bell-shaped with pKa values of 7.7 ± 0.3 and 8.6 ± 0.4. Km values for substrate increased with pH approximately 70-fold across the pH range 5.8−9.1. The Km for the H76N mutant was similar to that observed for native enzyme at high pH and was relatively constant across this pH range. The basic limb of the pH−rate profile is reduced but not abolished in the H76N mutant reaction. The results are discussed in terms of the possible role of His-76 and the nature of the transition state for catalysis in the native enzyme and mutant

Identification of the High Affinity Mn²⁺ Binding Site of Bacteriophage λ Phosphoprotein Phosphatase: Effects of Metal Ligand Mutations on Electron Paramagnetic Resonance Spectra and Phosphatase Activities^†

Bacteriophage λ phosphoprotein phosphatase (λPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of λPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to λPP are E. coli PrpA and PrpB. Using the crystal structure of λPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365−15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of λPP reconstituted with Mn2+ by electron paramagnetic resonance (EPR) spectroscopy, Mn2+ binding analysis, and phosphatase kinetics. Analysis of Mn2+-bound active site mutant λPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn2+ and [(Mn2+)2] binding. The high affinity Mn2+ binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site

Formation of a Stable Cyano-Bridged Dinuclear Iron Cluster Following Oxidation of the Superoxide Reductases from <i>Treponema </i><i>p</i><i>allidum</i> and <i>Desulfovibrio </i><i>v</i><i>ulgaris</i> with K₃Fe(CN)₆^‡

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K3Fe(CN)6 oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K3Fe(CN)6 reveals a band in the near-IR with λmax at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025−2030, and 2047 cm-1, assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe3+ ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site