Site Of Purple Acid Phosphatases Research Articles

Divalent metal ion in the active site of purple acid phosphatase modulates substrate binding: Kinetic and thermodynamic properties

The purple acid phosphatase was purified from 5.9-fold to apparent homogeneity from Anagelis arvensis seeds using SP-Sephadex C-50 and Sephadex G-100 chromatography. The results of residual activity tests conducted using different temperature ranges (50–70 °C) were calculated as the activation energy (Ed = 72 kJ/mol), enthalpy (69.31 ≤ (ΔH° ≤ 69.10 kJ/mol), entropy (−122.48 ≤ ΔS° ≤ −121.13 J/mol·K), and Gibbs free energy (108.87 ≤ ΔG° ≤ 111.25 kJ/mol) of the enzyme irreversible denaturation. These thermodynamic parameters indicate that this novel PAP is highly thermostable and may be significant for use in industrial applications. However, it may be confirmed by stopped-flow measurements that this substitution produces a chromophoric Fe3+ site and a Pi-substrate interaction that is about ten times faster. Additionally, these data show that phenyl phosphate hydrolysis proceeds more rapidly in metal form of A. arvensis PAP than the creation of a μ-1,3 phosphate complex. The Fe3+ site in the native Fe3+-Mn2+ derivative interacts with it at a faster rate than in the Fe3+-Fe2+ form. This is most likely caused by a network of hydrogen bonds between the first and second coordination spheres. This suggests that the choice of metal ions plays a significant role in regulating the activity of this enzyme.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24–40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26–28, 30, 33–36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

Biomimetics for purple acid phosphatases: A historical perspective

Biomimetics hold potential for varied applications in biotechnology and medicine but have also attracted particular interest as benchmarks for the functional study of their more complex biological counterparts, e.g. metalloenzymes. While many of the synthetic systems adequately mimic some structural and functional aspects of their biological counterparts the catalytic efficiencies displayed are mostly far inferior due to the smaller size and the associated lower complexity. Nonetheless they play an important role in bioinorganic chemistry. Numerous examples of biologically inspired and informed artificial catalysts have been reported, designed to mimic a plethora of chemical transformations, and relevant examples are highlighted in reviews and scientific reports. Herein, we discuss biomimetics of the metallohydrolase purple acid phosphatase (PAP), examples of which have been used to showcase synergistic research advances for both the biological and synthetic systems. In particular, we focus on the seminal contribution of our colleague Prof. Ademir Neves, and his group, pioneers in the design and optimization of suitable ligands that mimic the active site of PAP.

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: Avenues for improved phosphorus acquisition in crops

Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. Crystal structures of this enzyme in complex with ATP analogues (to 2.20 and 2.60 Å resolution, respectively) complement the recent structure of rkbPAP with a bound ADP analogue (ChemBioChem 20 (2019) 1536). Together these complexes provide the first structural insight of a PAP in complex with molecules that mimic biologically relevant substrates. Homology modeling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and thale cress (AtPAP26) that are efficient in hydrolyzing phytate and PEP as preferred substrates, respectively. The combining of crystallographic data, substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus) resulted in the identification of several active site residues that are important in defining the substrate specificities of plant PAPs; of particular relevance is the identification of a motif (“REKA”) that is characteristic for plant PAPs that possess phytase activity. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus acquisition and use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.

Theoretical studies on the mechanism of activation of phosphoprotein phosphatases and purple acid phosphatases suggest an evolutionary strategy to survive in acidic environments

Dephosphorylation reactions of phosphoprotein phosphatases (PPPs) share a common catalytic cycle. In one stage of the cycle, the active site is regenerated through formation of a new nucleophilic μ-hydroxy moiety and reprotonation of the proton donor, His125 (numbered according to the protein phosphatase 1 sequence). To date the exact details of the mechanism of this step remain uncertain. On the basis of recurring observations in several crystal structures, we propose an activation mechanism in which dephosphorylation of PPPs proceeds mainly through proton transfer from the water molecule that bridges the metal ions to His125, which is mediated by another water molecule. Our calculations using hybrid density functional theory and B3LYP functionals support this activation mechanism. We also propose that Asp95 facilitates proton transfer by eliminating the energy barrier and the backbone carbonyl oxygen atom of His248 acts mainly to orient and stabilize the μ-hydroxo (or water molecule) through hydrogen bonding. Furthermore, on the basis of the structural similarities of the active sites of purple acid phosphatases (PAPs) and PPPs, we speculate that PAPs are activated by a dual proton transfer mediated by one water molecule. Our calculations support this hypothesis and indicate that the active site of PAPs can still be active in an acidic environment (in agreement with the acid phosphatase activity of PAPs). Therefore, the variant of the activation mechanism from PPPs to PAPs implies an evolutionary adaptation to acidic environments.

Second-Coordination-Sphere Effects Increase the Catalytic Efficiency of an Extended Model for FeIIIMII Purple Acid Phosphatases

Herein we describe the synthesis of a new heterodinuclear Fe(III)Cu(II) model complex for the active site of purple acid phosphatases and its binding to a polyamine chain, a model for the amino acid residues around the active site. The properties of these systems and their catalytic activity in the hydrolysis of bis(2,4-dinitrophenyl)phosphate are compared, and conclusions regarding the effects of the second coordination sphere are drawn. The positive effect of the polymeric chain on DNA hydrolysis is also described and discussed.

Theoretical studies on the reaction mechanism of PP1 and the effects of different oxidation states of the Mn–Mn center on the mechanism

Protein phosphatase 1 (PP1) is a dinuclear metalloenzyme that catalyzes the dephosphorylation of serine and threonine residues. In this work, the catalytic reaction mechanism of PP1 was theoretically investigated by hybrid density functional theory. Firstly, an initial model of the Mn(II)-Mn(II) active site of PP1 was constructed on the basis of the high-resolution crystal structure, and stationary points along the reaction pathway were optimized and analyzed. The calculations provide strong support for the mechanism of the dephosphorylation by PP1 and suggest that His125 plays the role of donating a proton to the leaving group. Furthermore, reaction models with the Mn-Mn centers at different oxidation states [Mn(III)-Mn(II) and Mn(III)-Mn(III) centers] were designed. Our calculations show that increasing the oxidation state of one or both Mn(II) can shorten the bond lengths between the metal ions and the ligands, and increase the energy barrier of the related reactions. We found it interesting that artificially adding a negatively charged hydroxy ligand into the Mn(III)-Mn(II) center can recover the shortened coordination bonds and lower the increased energy barrier. Our investigation suggests that the definite oxidation states of the metal centers should be significantly correlated to the negative charges of the ligands not only in phosphoprotein phosphatases, but also in purple acid phosphatases and Escherichia coli 5'-nucleotidase. This means that all the members of phosphoprotein phosphatases adopt homodivalent centers, and suggests the heterovalent active sites of purple acid phosphatases have evolved from homodivalent ones.

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)-M(II) (MisFe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and divalent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H(3)HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe(4)(HPBA)(2)(μ-CH(3)COO)(2)(μ-O)(μ-OH)(OH(2))(2)]ClO(4)·5H(2)O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bell-shaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k (cat) of 1.6 (±0.2)×10(-3)s(-1), three kinetically relevant pK (a) values of 5.3, 6.2 and 8.4, with K (M) of 7.4±0.6mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.

Hydrolysis of phosphodiesters by diiron complexes: design of nonequivalent iron sites in purple acid phosphatase models.

New mu-oxo-diferric complexes have been designed for hydrolysis of phosphodiesters. To mimic the diiron active site of purple acid phosphatase, a combinatorial method has been used to select complexes containing two distinct iron coordination spheres. The introduction of a bidentate ligand, a substituted phenanthroline (L) into complex 1, [Fe2O(bipy)4(OH2)2](NO3)4, generates in solution the complex [Fe2O(bipy)3(L)(OH2)2](NO3)4 as shown by ESI/MS and 1H NMR studies. The latter complex was found to be 20-fold more active than complex 1. On the basis of kinetic studies, we demonstrated that the complex [Fe2O(bipy)3(L)(OH)(OH2)](NO3)3 was the active species and the reaction proceeded through the formation of a ternary complex in which one iron binds a hydroxide and the second, the substrate. At nonsaturating concentrations of the substrate, the increased activity with increased methyl substituents in L was due to an increased affinity of the complex for the substrate. The activity of [Fe2O(bipy)3(33'44'Me2-Phen)(OH2)2](NO3)4 [33'44'Me2Phen = 3,3',4,4'-dimethyl-1,10-phenanthroline] was found to be comparable to that reported for Co(III) or Ce(IV) complexes.

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism.

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.

A new bis(μ-alkoxo) diiron(III) complex and its implications regarding the number of Fe(III)–phenolate bonds and the redox potential in uteroferrin

The first diiron(III) complex of the ligand N,N′-bis(2-hydroxybenzyl)-2-hydroxypropane-1,3-diamine (H3bbpnol), [Fe2(bbpnol)2]·2H2O 1, was prepared and fully characterized in order to model diiron metalloenzymes. The X-ray crystal structure determination together with the electrochemical data of 1 gave us sufficient information to infer the number of phenolates terminally bonded to the iron(III) centers in the active sites of Purple Acid Phosphatases (PAPs). Each iron(III) center in complex 1 has two O-phenolate atoms terminally coordinated. By comparison of redox potentials for the process FeIII2–FeIIIFeII of the diiron PAP uteroferrin, complex 1 and complexes containing one, two and no phenolate oxygen atoms terminally bonded to the iron(III) centers, we confirmed the presence of only one terminal tyrosine group coordinated to the non-reducible iron(III) center, in agreement with the proposed structure of such metalloenzymes. The mixed-valence FeIIFeIII derivative of 1 has been characterized by spectroelectrochemistry in the UV–VIS region. The E°′ value obtained from the Nernst plot is in good agreement with CV data, and solutions of the FeIIFeIII species are stable under argon on the time scale of the spectropotentiostatic experiment. Magnetic susceptibility measurements in the range 4.5 to 300 K indicate very weak antiferromagnetic coupling between the iron ions with J = −2.2 cm−1, consistent with the long Fe–O distance and the small Fe–O–Fe angle within the bis(μ-alkoxo)diiron(III) entity.

Signal peptide sequence processing site of purple acid phosphatase from kidney bean (Phaseolus vulgaris L. Ohfuku) seeds.

We purified purple acid phosphatase (PAPase) from kidney bean (Phaseolus vulgaris L. Ohfuku) seeds, a Japanese cultivar of the kidney bean. The molecular mass of the purified native enzyme was estimated approximately 110kDa by gel filtration. Following SDS-PAGE in the presence of 2-mercaptoethanol, glycosylated polypeptides of major 61kDa and minor 59kDa were observed. The N-terminal amino acid sequences for both bands were determined to be GKSSNFVRKTNKNRDMPLDS, suggesting they were two different subunits with the same N-terminal but probably different C-terminal or degree of glycosylation. The partial nucleotide sequences covering the N-terminal region were also determined using polymerase chain reaction (PCR). Comparison with the deduced amino acid sequence from the nucleotide sequence and that of the purified enzyme revealed that the N-terminal amino acid residue corresponded to Gly 23 from the initiation colon (Met). For the deduced amino acid sequence of the PAPase gene, a SignalP (ver. 2.0) analysis program predicted that a 22 amino acid sequence at the N-terminus of a precursor protein was signal peptide. This prediction was in agreement with the fact that PAPase with a signal peptide at the N-terminus was cleaved between Gly 22 and Gly 23 by putative signal peptidase.

EXAFS studies on the active site of purple acid phosphatase from sweet potato Ipomoea batatas.

XAS investigations on the active site of the purple acid phosphatase isolated from the sweet potato Ipomoea batatas provided first information about the structure. Data analysis reveal a dinuclear active site containing an iron and a zinc atom. The first coordination sphere of each metal in the active site consists of six nitrogen/oxygen donor atoms, resulting in a distorted octahedral coordination sphere very similar to the active site of the enzyme isolated from the red kidney bean Phaseolus vulgaris. The XAS spectra were recorded in fluorescence mode at 18 K using a Canberra 13-element germanium solid state detector.

Carboxylate/Diphenylphosphate Exchanges in Asymmetric Diiron Complexes Modeling the Purple Acid Phosphatases Enzymes: Associated Redox Processes

Redox effects dominate the carboxylate/diphenylphosphate ligand exchange in diiron complexes which model the active site of purple acid phosphatases.

Dinuclear iron (III) complex with a bridging diphenylphosphate and two terminal diphenylphosphate ligands

Novel dinuclear iron (III) complex [Fe2(HPTB) 1μ-O2P(OPh)2 O2P(OPh)2 2] (C1O4)2, where HPTB is the anion of N, N, N′, N′-tetrakis(2-benzimidawlylmethyl)-2-hydroxy-1, 3-diaminopropane, was prepared in order to provide a model for the active site of purple acid phosphatase (PAP). This compound has been characterized by X-ray crystallography. Crystal data: triclinic, space group\(P \bar 1\),a = 1. 512 13(5), b = 1. 591 19(5),c = 1.890 90(5) nm, α = 69.925(1)°,β = 84. 358(1)°,γ = 65.712(1)°, Z = 2. Dinuclear iron (II1) centers are bridged by aμ-OR group from HPTB, a bidentate bridging diphenylphosphate and two terminal diphenylphosphates coordinate to Fe(III) centers.

Modelando a coordenação de fosfato com o sítio ativo das fosfatases ácidas púrpuras

We present a new binuclear complex, Fe2III(BBPMP)(OH)(O2 P(OPh)2) ClO4.CH3OH, 3, where BBPMP is the anion of 2,6-bis(2-hydroxybenzyl)(2-pyridylmethyl) aminomethyl-4-methylphenol, as a suitable model for the chromophoric site of purple acid phosphatases coordinated to phosphate. The complex was obtained by the reaction of complex 2, Fe2III(BBPMP)(O2P(OPh) 2)2 ClO4.H2O, in CH3CN with one equivalent of triethylamine. Based on the chromophoric properties of the model complex, lmax = 560 nm/ e = 4480 M-1 cm-1/Fe2 compared to the enzyme coordinated to phosphate, we can speculate about a possible mechanism of fixing this oxyanion by the oxidized form of the enzymes.

A new asymmetric N 5O 2-donor binucleating ligand and its first Fe IIFe III complex as a model for the redox properties of uteroferrin

A new N,O-donor binucleating ligand, 2-bis[{(2-pyridylmethyl)-aminomethyl}-{(2-hydroxybenzyl)(2-pyridylmethyl)}-aminomethyl]-4-meth (H 2BPBPMP), and its first binuclear Fe IIFe III complex have been prepared in order to provide synthetic analogues for the coordination environment on the active site of purple acid phosphatases. The complex [Fe IIFe III(BPBPMP)(OAc) 2]ClO 4·CH 3OH was obtained from a self-assembled type reaction between Fe(ClO 4) 2· 6H 2B, H 2BPBPMP and sodium acetate trihydrate in methanol. The complex has been characterized by IR, CHN, molar conductivity, magnetic susceptibility, Mössbauer, electronic absorption spectroscopies and electrochemical methods of analyses, and turns out to be the first synthetic analogue for the redox properties ( E°′=0.38 V versus NHE) of uteroferrin.

Magnetic investigations on the active site of purple acid phosphatase from kidney-beans (PAP), a 2Fe-enzyme

Magnetic investigations on the active site of purple acid phosphatase from kidney-beans (PAP), a 2Fe-enzyme

Synthesis, Crystal Structure, and Properties of a Polyimidazole Diiron(III) Complex. A New Model of the Active Site of Purple Acid Phosphatase

Synthesis, Crystal Structure, and Properties of a Polyimidazole Diiron(III) Complex. A New Model of the Active Site of Purple Acid Phosphatase

A Mixed‐Valent, Unsymmetrical FeIIFeIII Complex with a Terminal Phenolato Ligand as a Model for the Active Site of Purple Acid Phosphatases

The structure and the magnetic and electrochemical properties of the active center of purple acid phosphatases are modeled by a complex that mimics the terminal coordination of the tyrosine residue and was prepared with a new heptadentate phenola‐to ligand system. The instability of the reduced FeIIFeII state of the enzyme may be attributed to the terminal ligand, since the phenolato ligand in the model is a destabilizing factor.