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Gerhard Schenk - One of the best experts on this subject based on the ideXlab platform.
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structural elements that modulate the substrate specificity of plant Purple Acid Phosphatases avenues for improved phosphorus acquisition in crops
bioRxiv, 2019Co-Authors: Daniel Feder, Ross P. Mcgeary, Luke W Guddat, Natasa Mitic, Thierry G A Lonhienne, Agnelo Furtado, B Schulz, Robert J Henry, Susanne Schmidt, Gerhard SchenkAbstract:Abstract 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. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 A resolution) provides insight into the amino Acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus 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.
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asymmetric mono and dinuclear gaiii and znii complexes as models for Purple Acid Phosphatases
Journal of Inorganic Biochemistry, 2016Co-Authors: Peter Comba, Simone Bosch, Lawrence R Gahan, Gerhard SchenkAbstract:Derivatives of the known dinucleating ligands HL1 (2,6-bis{[bis(pyridin-2-ylmethyl)amino]methyl}-4-methylphenol) and H2L2 (2-{[bis(pyridin-2-ylmethyl)amino]methyl}-6-{[(2-hydroxybenzyl)(pyridine-2-ylmethyl)amino]methyl}-4-methylphenol) with two pivaloylamido hydrogen bond donor substituents, H3L3 and H3L5, have been prepared. The mono-, homo- and heterodinuclear ZnII and GaIII complexes of these ligands have been prepared and characterized. The solution equilibria are discussed on the basis of extensive NMR spectroscopic, mass spectrometric and pH-dependent UV-vis spectroscopic titrations. The phosphoester hydrolysis activity of the complexes has been studied as a function of pH and substrate concentration and analyzed using Michaelis-Menten kinetics. It emerges that the mixed metal (mixed valent) complex of the ligand with an asymmetric disposition of the hydrogen bonding substituents (H3L3) is a functional model for the mixed valent, dinuclear metallohydrolase Purple Acid phosphatase. This complex combines the essential structural features of the active site of PAP and is the first heterodinuclear model complex mimicking the essential function of PAPs, i.e. the hydrolysis of phosphomonoesters.
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an approach to more accurate model systems for Purple Acid Phosphatases paps
Inorganic Chemistry, 2015Co-Authors: Paul V Bernhardt, Gerhard Schenk, Simone Bosch, Peter Comba, Lawrence R Gahan, Graeme R Hanson, Valeriu Mereacre, Christopher J Noble, Annie K Powell, Hubert WadepohlAbstract:The active site of mammalian Purple Acid Phosphatases (PAPs) have a dinuclear iron site in two accessible oxidation states (FeIII2 and FeIIIFeII), and the heterovalent is the active form, involved in the regulation of phosphate and phosphorylated metabolite levels in a wide range of organisms. Therefore, two sites with different coordination geometries to stabilize the heterovalent active form and, in addition, with hydrogen bond donors to enable the fixation of the substrate and release of the product, are believed to be required for catalytically competent model systems. Two ligands and their dinuclear iron complexes have been studied in detail. The solid-state structures and properties, studied by X-ray crystallography, magnetism, and Mossbauer spectroscopy, and the solution structural and electronic properties, investigated by mass spectrometry, electronic, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Mossbauer spectroscopies and electrochemistry, are discussed in detai...
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the applications of binuclear metallohydrolases in medicine recent advances in the design and development of novel drug leads for Purple Acid Phosphatases metallo β lactamases and arginases
ChemInform, 2014Co-Authors: Ross P. Mcgeary, Gerhard Schenk, Luke W GuddatAbstract:Binuclear metallohydrolases are a family of proteins that can be targeted for drug discovery. The common feature of these enzymes is the presence of two closely spaced metal ions (i.e. less than 4 angstrom apart) that capture a water molecule that is used as a nucleophile in highly specific hydrolytic reactions. In this mini-review we describe what is known about the biological and catalytic activity, three-dimensional structure and inhibition for three prominent drug targets in this family of enzymes, (i) Purple Acid Phosphatases, (ii) metallo-beta-lactamases and (iii) arginases. These enzymes are targets for the development of chemotherapeutics to treat a range of disorders including osteoporosis, cardiovascular disease and erectile dysfunctions, but also to stem the spread of antibiotic resistance, a major threat to global health care. (C) 2014 Elsevier Masson SAS. All rights reserved.
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Purple Acid phosphatase a journey into the function and mechanism of a colorful enzyme
Coordination Chemistry Reviews, 2013Co-Authors: Gerhard Schenk, Graeme R Hanson, Natasa Mitic, Peter CombaAbstract:Abstract Purple Acid Phosphatases (PAPs) catalyze the hydrolysis of a wide range of phosphomonoester and amide substrates. These enzymes have been identified and characterized from numerous plant and animal sources, and it is likely that a limited number of bacterial organisms also utilize this catalyst. The biological roles of this enzyme are diverse, including bone resorption, microbial killing and possibly iron transport in animals, and phosphate acquisition in plants. While animal and plant PAPs share less than 20% amino Acid sequence identity and differ (with a couple of exceptions) greatly in size (35 kDa vs. 55 kDa per monomer) and oligomeric structure (monomer vs. homodimer), their catalytically relevant active sites are highly conserved, with seven invariant amino Acid side chains coordinating an Fe 3+ and an M 2+ (M = Fe or Zn, Mn in animal or plant PAPs, respectively). Recent functional studies have indicated that PAPs are rather flexible in terms of the precise mechanistic strategy they may employ. Here, we review advances that have facilitated detailed insight into how these enzymes operate. The knowledge gained is not only of interest for coordination chemists and biochemists who focus on the physicochemical and mechanistic properties of the active site metal ion center in a metalloenzyme, but also for medicinal chemists who aim to exploit PAP as a target for the development of novel chemotherapeutics to treat osteoporosis.
Ademir Neves - One of the best experts on this subject based on the ideXlab platform.
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a new heteropentanuclear complex containing the fe2iiizn3ii μ oh 3 structural motif as a model for Purple Acid Phosphatases
Inorganica Chimica Acta, 2020Co-Authors: Filipy Gobbo Maranha, Adailton J. Bortoluzzi, Rosely A. Peralta, Ebbe Nordlander, Graciela Aparecida Dos Santos Silva, Ademir NevesAbstract:Herein, we describe the synthesis and X-ray structure of a new heteropentanuclear complex (2) containing a [Fe2 IIIZn3 II(μ-OH)3] structural unit and the unsymmetrical ligand H2L2-et. The molecular structure of (2) shows that it is formed by a basic dinuclear [FeIII(μ-OH)ZnII(L2-et)] unit that is connected to a second dinuclear [FeIII(μ-OH)ZnII(L2-et)] unit through a hydroxo bridge while a third ZnII ion is coordinated by the pendant 1,2-ethanediamine groups of H2L2-et, resulting in the pentanuclear complex. Kinetic studies on the hydrolysis of the substrate 2,4-BDNPP (bis(2,4-dinitrophenyl)phosphate) reveal that (2) shows diesterase activity. While the kinetic activity is comparable to the corresponding dinuclear FeIIIZnII complex containing the same ligand, the association with 2,4-BDNPP is significantly decreased. (Less)
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Second-Coordination-Sphere Effects Increase the Catalytic Efficiency of an Extended Model for FeIIIMII Purple Acid Phosphatases
Inorganic chemistry, 2013Co-Authors: Bernardo De Souza, Gabriel L. Kreft, Tiago Bortolotto, Hernán Terenzi, Adailton J. Bortoluzzi, Eduardo E. Castellano, Rosely A. Peralta, Josiel B. Domingos, Ademir NevesAbstract:Herein we describe the synthesis of a new heterodinuclear FeIIICuII 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.
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a synthetic pathway for an unsymmetrical n5o2 heptadentate ligand and its heterodinuclear iron iii zinc ii complex a biomimetic model for the Purple Acid Phosphatases
Chemistry & Biodiversity, 2012Co-Authors: Adailton J. Bortoluzzi, Fernando R Xavier, Ademir NevesAbstract:One major field of interest in bioinorganic chemistry is the design and synthesis of inorganic compounds with low molecular mass, showing structural, spectroscopic, and reactivity properties that mimic enzymes, such as Purple Acid Phosphatases (PAPs). In this study, the unsymmetrical heptadentate ligand 2-[(4,7-diisopropyl-1,4,7-triazacyclonon-1-yl)methyl]-6-{[(2-hydroxybenzyl)(pyridin-2-ylmethyl)-amino]methyl}-4-methylphenol (H(2)L) and its first mixed-valence complex [Fe(III)Zn(II)(L)(μ-OAc)(2)]ClO(4)(1) were synthesized. Physical and chemical measurements (crystal structure, conductometry, IR and UV/VIS spectroscopy, and electrochemistry) were performed for 1, and these properties are compared with those presented by the kbPAPs active sites. Potentiometric titration studies of 1 have confirmed its Acid/base properties that are crucial for the understanding of the phosphodiester and DNA catalytic cleavage in future studies.
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broken symmetry density functional study of biomimetic models for Purple Acid Phosphatases of the type fe iii m ii m fe cu ni co and mn
Computational and Theoretical Chemistry, 2012Co-Authors: Dalva E C Ferreira, Ademir Neves, Wagner B De Almeida, Willian R RochaAbstract:Abstract Density Functional Theory (DFT) calculations using the hybrid meta-GGA TPSSh functional, were carried out for mixed valence compounds Fe(III)– M (II) ( M = Fe, Cu, Ni, Co and Mn) which are structural and functional biomimetic models for Purple Acid Phosphatases. The broken symmetry (BS) formalism was used to compute the magnetic coupling constant which shows that all complexes exhibit weak antiferromagnetic coupling. The effect of the geometry on the accuracy of the BS calculations is evaluated and it is shown that the TPSSh functional provides accurate results with errors in the range of 10–19%, as long as the geometry around the transition metal ions is well described. All complexes show one magnetic exchange σ/π pathway involving the in-plane p -orbitals of the phenoxo bridge and the metal d -orbitals. Additionally, a correlation between the computed coupling constant and the M II –O(bridge) bond distance is found, in agreement with the experimental findings.
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unsymmetrical feiiicoii and gaiiicoii complexes as chemical hydrolases biomimetic models for Purple Acid Phosphatases paps
Inorganic Chemistry, 2009Co-Authors: Fernando R Xavier, Hernán Terenzi, Adailton J. Bortoluzzi, Rosely A. Peralta, Ademir Neves, Annelise Casellato, Bruno Szpoganicz, Patricia Cardoso Severino, Z Tomkowicz, Sergei OstrovskyAbstract:The design and development of suitable biomimetic catalytic systems capable of mimicking the functional properties of enzymes continues to be a challenge for bioinorganic chemists. In this study, we report on the synthesis, X-ray structures, and physicochemical characterization of the novel isostructural [(FeCoII)-Co-III(BPBPMP)(mu-OAc)(2)]ClO4 (1) and [(GaCoII)-Co-III(BPBPMP)(mu-OAc)(2)]ClO4 (2) complexes with the unsymmetrical dinucleating ligand H2BPBPMP {2-bis[{(2-pyridyl-methyl)-aminomethyl)-6-{(2-hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methylphenol). The previously reported complex [(FeZnII)-Zn-III(BPBPMP)(mu-OAc)(2)]ClO4 (3) was investigated here by electron paramagnetic resonance for comparison with such studies on 1 and 2. A magneto-structural correlation between the exchange parameter J (cm(-1)) and the average bond lengh d (angstrom) of the [Fe-III-O-M-II] structural unit for 1 and for related isostructural (FeMII)-M-III complexes using the correlation J = -10(7) exp(-6.8d) reveals that this parameter is the major factor that determines the degree of antiferromagnetic coupling in the series [(BPBPMP)Fe-III(mu-OAc)(2)M-II](+) (M-II = Mn, Fe, Co, Ni) of complexes. Potentiometric and spectrophotometric titrations along with electronic absorption studies show that, in aqueous solution, complexes 1 and 2 generate the [(HO)M-III(mu-OH)Co-II(H2O)] complex as the catalytically active species in diester hydrolysis reactions, Kinetic studies on the hydrolysis of the model substrate bis(2,4-dinitrophenyl)phosphate by 1 and 2 show Michaelis-Menten behavior, with 2 being 35% more active than 1. In combination with k(H)/k(D) isotope effects, the kinetic studies suggest a mechanism in which a terminal M-III-bound hydroxide is the hydrolysis-initiating nucleophilic catalyst. In addition, the complexes show maximum catalytic activity in DNA hydrolysis near physiological pH. The modest reactivity difference between 1 and 2 is consistent with the slightly increased nucleophilic character of the Ga-III-OH terminal group in comparison to Fe-III-OH in the dinuclear (MCoII)-Co-III species.
Lawrence R Gahan - One of the best experts on this subject based on the ideXlab platform.
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asymmetric mono and dinuclear gaiii and znii complexes as models for Purple Acid Phosphatases
Journal of Inorganic Biochemistry, 2016Co-Authors: Peter Comba, Simone Bosch, Lawrence R Gahan, Gerhard SchenkAbstract:Derivatives of the known dinucleating ligands HL1 (2,6-bis{[bis(pyridin-2-ylmethyl)amino]methyl}-4-methylphenol) and H2L2 (2-{[bis(pyridin-2-ylmethyl)amino]methyl}-6-{[(2-hydroxybenzyl)(pyridine-2-ylmethyl)amino]methyl}-4-methylphenol) with two pivaloylamido hydrogen bond donor substituents, H3L3 and H3L5, have been prepared. The mono-, homo- and heterodinuclear ZnII and GaIII complexes of these ligands have been prepared and characterized. The solution equilibria are discussed on the basis of extensive NMR spectroscopic, mass spectrometric and pH-dependent UV-vis spectroscopic titrations. The phosphoester hydrolysis activity of the complexes has been studied as a function of pH and substrate concentration and analyzed using Michaelis-Menten kinetics. It emerges that the mixed metal (mixed valent) complex of the ligand with an asymmetric disposition of the hydrogen bonding substituents (H3L3) is a functional model for the mixed valent, dinuclear metallohydrolase Purple Acid phosphatase. This complex combines the essential structural features of the active site of PAP and is the first heterodinuclear model complex mimicking the essential function of PAPs, i.e. the hydrolysis of phosphomonoesters.
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an approach to more accurate model systems for Purple Acid Phosphatases paps
Inorganic Chemistry, 2015Co-Authors: Paul V Bernhardt, Gerhard Schenk, Simone Bosch, Peter Comba, Lawrence R Gahan, Graeme R Hanson, Valeriu Mereacre, Christopher J Noble, Annie K Powell, Hubert WadepohlAbstract:The active site of mammalian Purple Acid Phosphatases (PAPs) have a dinuclear iron site in two accessible oxidation states (FeIII2 and FeIIIFeII), and the heterovalent is the active form, involved in the regulation of phosphate and phosphorylated metabolite levels in a wide range of organisms. Therefore, two sites with different coordination geometries to stabilize the heterovalent active form and, in addition, with hydrogen bond donors to enable the fixation of the substrate and release of the product, are believed to be required for catalytically competent model systems. Two ligands and their dinuclear iron complexes have been studied in detail. The solid-state structures and properties, studied by X-ray crystallography, magnetism, and Mossbauer spectroscopy, and the solution structural and electronic properties, investigated by mass spectrometry, electronic, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Mossbauer spectroscopies and electrochemistry, are discussed in detai...
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phosphate ester cleavage promoted by a tetrameric iron iii complex
Journal of Biological Inorganic Chemistry, 2011Co-Authors: Anob Kantacha, Gerhard Schenk, Sarah J Smith, Rebecca R Buchholz, Lawrence R GahanAbstract:The Purple Acid Phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)-M(II) (M is Fe, 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)(mu-CH(3)COO)(2)(mu-O)(mu-OH)(OH(2))(2)]ClO(4)center dot 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) x 10(-3) s(-1), three kinetically relevant pK(a) values of 5.3, 6.2 and 8.4, with K(M) of 7.4 +/- 0.6 mM. 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.
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electronic structure and spectro structural correlations of feiiiznii biomimetics for Purple Acid Phosphatases relevance to dna cleavage and cytotoxic activity
Inorganic Chemistry, 2010Co-Authors: Rosely A. Peralta, Bernardo De Souza, Adailton J. Bortoluzzi, Rafael Jovito, Fernando R Xavier, Ricardo A A Couto, Annelise Casellato, Faruk Nome, Andrew Dick, Lawrence R GahanAbstract:Purple Acid Phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear FeIIIMII center (MII = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric Acid esters. The dinuclear complex [(H2O)FeIII(μ-OH)ZnII(L-H)](ClO4)2 (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N′-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H2L-H) has recently been prepared and is found to closely mimic the coordination environment of the FeIIIZnII active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the FeIII center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted FeIIIN2O4 octahedron with a dominant tetragonal compression aligned along the μ-OH−Fe...
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the divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis
Journal of the American Chemical Society, 2010Co-Authors: Natasa Mitic, Lawrence R Gahan, Alvan C Hengge, Kieran S Hadler, Gerhard SchenkAbstract:The Purple Acid Phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe(3+)-Fe(2+) pig PAP (uteroferrin; Uf) and its Fe(3+)-Mn(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe(2+) by Mn(2+) lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a mu-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe(3+) site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe(3+). The faster rates of interaction with the Fe(3+) site in the Fe(3+)-Mn(2+) derivative than the native Fe(3+)-Fe(2+) form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.
William C. Plaxton - One of the best experts on this subject based on the ideXlab platform.
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Phosphoprotein Phosphatase Function of Secreted Purple Acid Phosphatases
Protein Phosphatases and Stress Management in Plants, 2020Co-Authors: Mina Ghahremani, William C. PlaxtonAbstract:Purple Acid Phosphatases (PAPs) are metalloenzymes that hydrolyze orthophosphate (Pi) from Pi monoesters with an Acidic pH optimum. Plant PAPs belong to a relatively large gene family whose respective functions remain poorly understood. However, it has been well established that certain Pi-starvation-inducible vacuolar PAP isozymes remobilize Pi from expendable intracellular Pi monoesters and anhydrides during nutritional Pi deficiency, a common abiotic stress that plants are subjected to in their natural environment. By contrast, several different PAPs are upregulated and targeted to the extracellular matrix (ECM) during Pi deprivation where they scavenge and recycle Pi from organic P compounds present in the rhizosphere or that have leaked from the cytoplasm into cell walls and the apoplast. The aim of this chapter is to provide a brief overview of the central role of PAPs in plant Pi acquisition and use efficiency, followed by a discussion of evidence for (1) extracellular protein phosphorylation networks in the animal and plant kingdoms and (2) phosphoprotein phosphatase function of certain secreted PAP isozymes. It has been well established that HsACP5, the single low molecular weight (35 kDa) PAP encoded by the human genome, plays a pivotal role in bone metabolism by dephosphorylating osteopontin, a secreted bone matrix phosphoprotein. Similarly, several secreted plant PAP isozymes appear to function in signaling and/or cell wall metabolism by dephosphorylating endogenous ECM phosphoproteins. Establishing the impact of development and stress on the extracellular phosphoproteome of plant cells, the occurrence and targets of secreted plant protein kinases, as well as the phosphoprotein phosphatase roles of secretory PAPs in planta promises to be a rewarding avenue for future studies.
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senescence inducible cell wall and intracellular Purple Acid Phosphatases implications for phosphorus remobilization in hakea prostrata proteaceae and arabidopsis thaliana brassicaceae
Journal of Experimental Botany, 2014Co-Authors: Michael W Shane, Kyla A Stigter, Eric T Fedosejevs, William C. PlaxtonAbstract:Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular Acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120 kDa and 60 kDa intracellular APase isoforms, whereas only the 120 kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120 kDa and 60 kDa APases partially purified from senescing leaves indicated that they are Purple Acid Phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55 kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual tar geting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested.
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biochemical and molecular characterization of atpap12 and atpap26 the predominant Purple Acid phosphatase isozymes secreted by phosphate starved arabidopsis thaliana
Plant Cell and Environment, 2010Co-Authors: Hue T Tran, Brenden A Hurley, Daowen Wang, Weiqiang Qian, Yimin She, William C. PlaxtonAbstract:Plant Purple Acid Phosphatases (PAPs) belong to a large multigene family whose specific functions in Pi metabolism are poorly understood. Two PAP isozymes secreted by Pi-deficient (−Pi) Arabidopsis thaliana were purified from culture filtrates of −Pi suspension cells. They correspond to an AtPAP12 (At2g27190) homodimer and AtPAP26 (At5g34850) monomer composed of glycosylated 60 and 55 kDa subunit(s), respectively. Each PAP exhibited broad pH activity profiles centred at pH 5.6, and overlapping substrate specificities. Concanavalin-A chromatography resolved a pair of secreted AtPAP26 glycoforms. AtPAP26 is dual targeted during Pi stress because it is also the principal intracellular (vacuolar) PAP up-regulated by −Pi Arabidopsis. Differential glycosylation appears to influence the subcellular targeting and substrate selectivity of AtPAP26. The significant increase in secreted Acid phosphatase activity of −Pi seedlings was correlated with the appearance of immunoreactive AtPAP12 and AtPAP26 polypeptides. Analysis of atpap12 and atpap26 T-DNA mutants verified that AtPAP12 and AtPAP26 account for most of the secreted Acid phosphatase activity of −Pi wild-type seedlings. Semi-quantitative RT-PCR confirmed that transcriptional controls exert little influence on the up-regulation of AtPAP26 during Pi stress, whereas AtPAP12 transcripts correlate well with relative levels of secreted AtPAP12 polypeptides. We hypothesize that AtPAP12 and AtPAP26 facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation.
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feeding hungry plants the role of Purple Acid Phosphatases in phosphate nutrition
Plant Science, 2010Co-Authors: Hue T Tran, Brenden A Hurley, William C. PlaxtonAbstract:Abstract Phosphate (Pi) is an essential, but limiting macronutrient that plays critical roles in plant metabolism and development. Plants have evolved an intricate array of adaptations to enhance Pi acquisition and utilization from their environment. The availability of the complete genome sequence of the model plant Arabidopsis thaliana , together with a wide assortment of related genomic resources, has significantly advanced our understanding of the adaptations of Pi-starved plants. Information on the genetic identity, subcellular location, biochemical properties, and probable functions of Acid Phosphatases involved in the Pi metabolism of Pi-starved Arabidopsis is beginning to emerge. Acid Phosphatases catalyze the hydrolysis of Pi from a broad range of phosphomonoesters with an Acidic pH optimum. The Arabidopsis genome encodes 29 different Purple Acid Phosphatases whose expression is influenced by various developmental and environmental factors. Pi starvation induces de novo synthesis of several extra- and intracellular Arabidopsis Purple Acid phosphatase isozymes; AtPAP12 and AtPAP26 appear to be the principal root-secreted Acid Phosphatases that scavenge Pi from extracellular Pi-esters, whereas the dual-targeted AtPAP26 is the predominant intracellular Acid phosphatase that functions in vacuolar Pi recycling by Pi-starved Arabidopsis . The identification and functional characterization of intracellular and secreted Purple Acid phosphatase isozymes upregulated by Pi-deprived plants may help develop strategies for engineering Pi-efficient crops, thereby minimizing the use of unsustainable Pi fertilizers in agriculture.
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purification and characterization of two secreted Purple Acid phosphatase isozymes from phosphate starved tomato lycopersicon esculentum cell cultures
FEBS Journal, 2002Co-Authors: Gale G Bozzo, Kashchandra G Raghothama, William C. PlaxtonAbstract:Two secreted Acid Phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells.SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-dayold Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 lmol Pi producedAEmin )1 mgAEprotein )1 , respectively.SDS/PAGE, periodic Acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are Purple Acid Phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by L-tartrate.The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs.CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct.Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable.Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1.SAP1 and SAP2 were activated by up to 80% by 5 mM Mg 2+ , and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively.Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg 2+ or molybdate.This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.
Bernt Krebs - One of the best experts on this subject based on the ideXlab platform.
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structure function relationships of Purple Acid phosphatase from red kidney beans based on heterologously expressed mutants
Archives of Biochemistry and Biophysics, 2005Co-Authors: Ngoc Thanh Truong, Joseph Itor Naseri, Andreas Vogel, Annette Rompel, Bernt KrebsAbstract:Purple Acid Phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino Acids, residing near the bimetallic active site. Based on the amino Acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.
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dinuclear zinc ii iron iii and iron ii iron iii complexes as models for Purple Acid Phosphatases
European Journal of Inorganic Chemistry, 2001Co-Authors: Sabine Albedyhl, Marie Therese Averbuchpouchot, Catherine Belle, Bernt Krebs, Jean Louis Pierre, Eric Saintaman, Stephane TorelliAbstract:The heterodinuclear ZnIIFeIII complex 1 and the isostructural FeIIFeIII complex 2 with the dinucleating ligand from 2,6-bis[{bis(2-pyridylmethyl)amino}methyl]-4-methoxyphenol (HBPMOP, 3) were prepared and characterized by X-ray crystallography. Solution studies (UV/Vis spectroscopy; electrochemistry) are described. A pH-induced change in the coordination spheres of the metal centers is seen. These complexes serve as models for the mixed-valence oxidation state in Purple Acid Phosphatases. The cleavage acceleration of the activated phosphodiester 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) was investigated in acetonitrile/water (1:1) in the presence of complexes of the ligand BPMOP and its methyl analogue BPMP with regards to its dependence on the pH value. At the optimum pH value (8.5 ± 0.2), the ZnIIFeIII complex from BPMOP shows a 2-fold higher rate acceleration compared with that of the complex containing BPMP. The diiron complex from BPMOP is 4-fold more reactive than the homologous complex from BPMP. The heterodinuclear ZnIIFeIII catalysts are at least 10-fold more reactive than the homonuclear FeIIFeIII catalysts.
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Structural and functional studies on model compounds of Purple Acid Phosphatases and catechol oxidases
Coordination Chemistry Reviews, 1999Co-Authors: Roberto Than, Arnold A. Feldmann, Bernt KrebsAbstract:The synthesis, single crystal X-ray crystallographic, magnetic and electrochemical characterization of eight representative symmetric and unsymmetric complexes as structural model compounds for active sites in PAPs is reported. A mixed valent diiron as well as an iron(III)–zinc(II) complex as models for the active, reduced form of mammalian and plant PAPs, respectively, were synthesized and characterized. Five diiron(III) compounds as structural models for the oxidized uteroferrin-phosphato and -arsenato complex and a model for the oxidized form of PAP from beef spleen are reported. In addition to the structural relevance the catalase and peroxidase activity of one of these model complexes is introduced. Further we summarize our recent research concerning synergistic investigations on catechol oxidase and on synthetic copper coordination complexes. The catechol oxidase is an important type 3 copper protein for the activation of dioxygen. The development of low-molecular weight catalysts should facilitate the oxidation of organic substances by O2. In particular the reported copper(II) complexes may serve as structural and functional bioinorganic model compounds for the active sites of dioxygen binding and dioxygen activating copper proteins, respectively. These investigations provided a new X-ray crystallographically characterized type of peroxo copper(II) complexes with a μ4-(η1)4 coordination mode.
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structural relationship between the mammalian fe iii fe ii and the fe iii zn ii plant Purple Acid Phosphatases
FEBS Letters, 1995Co-Authors: Thomas Klabunde, Bernt Krebs, Norbert Strater, Herbert WitzelAbstract:The primary structure of uteroferrin (Uf), a 35 kDa monomeric mammalian Purple Acid phosphatase (PAP) containing a Fe(III)-Fe(II) center, has been compared with the sequence of the homodimeric 111 kDa Fe(III)-Zn(II) kidney bean Purple Acid phosphatase (KBPAP). The alignment suggests that the amino Acid residues ligating the dimetal center are identical in Uf and KBPAP, although the geometry of the coordination sphere might slightly differ. Secondary structure predictions indicate that Uf contains two beta alpha beta alpha beta motifs thus resembling the folding topology of the plant enzyme. Guided by the recently determined X-ray structure of KBPAP a tentative model for the mammalian PAP can be constructed.
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crystal structure of a Purple Acid phosphatase containing a dinuclear fe iii zn ii active site
Science, 1995Co-Authors: Norbert Strater, Thomas Klabunde, Herbert Witzel, Paul Tucker, Bernt KrebsAbstract:Kidney bean Purple Acid phosphatase (KBPAP) is an Fe(III)-Zn(II) metalloenzyme resembling the mammalian Fe(III)-Fe(II) Purple Acid Phosphatases. The structure of the homodimeric 111-kilodalton KBPAP was determined at a resolution of 2.9 angstroms. The enzyme contains two domains in each subunit. The active site is located in the carboxyl-terminal domain at the carboxy end of two sandwiched beta alpha beta alpha beta motifs. The two metal ions are 3.1 angstroms apart and bridged monodentately by Asp164. The iron is further coordinated by Tyr167, His325, and Asp135, and the zinc by His286, His323, and Asn201. The active-site structure is consistent with previous proposals regarding the mechanism of phosphate ester hydrolysis involving nucleophilic attack on the phosphate group by an Fe(III)-coordinated hydroxide ion.
