Showing papers on "Purple acid phosphatases published in 2002"
TL;DR: The present work demonstrates that structure variation and expression regulation of plant PAPs are more complex than previously described and provides a framework for comprehensive molecular genetic and biochemical studies of all Arabidopsis P APs in the future.
268 citations
TL;DR: Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells and 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 ofextracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.
Abstract: 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.
149 citations
TL;DR: Solution studies of 1 indicate that a pH-induced change of the bridging acetate occurs, and the formation of an active [(OH)Fe(III)Zn(II)(OH(2))] species as a highly efficient catalyst under weakly acidic conditions for phosphate diesters hydrolysis is proposed.
Abstract: The new heterodinuclear complex [Fe(III)Zn(II)(BPBPMP)(OAc)(2)]ClO(4) (1) with the unsymmetrical N(5)O(2) donor ligand 2-bis[((2-pyridylmethyl)-aminomethyl)-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]-aminomethyl]-4-methylphenol (H(2)BPBPMP) has been synthesized and characterized by X-ray crystallography, which reveals that the complex cation has an Fe(III)Zn(II)(mu-phenoxo)-bis(mu-carboxylato) core. Solution studies of 1 indicate that a pH-induced change of the bridging acetate occurs, and the formation of an active [(OH)Fe(III)Zn(II)(OH(2))] species as a highly efficient catalyst under weakly acidic conditions for phosphate diesters hydrolysis is proposed.
104 citations
TL;DR: The present study shows 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.
Abstract: 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.
67 citations
TL;DR: The results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.
Abstract: Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the activ...
65 citations
TL;DR: The mechanism considered accounts for most but not all of the features displayed, and an inhibition process in which a second [H(2)PO(4)(-)] coordinates to the dinuclear center is proposed.
Abstract: The FeII of the binuclear FeIIFeIII active site of pig purple acid phosphatase (uteroferrin) has been replaced in turn by five MII ions (MnII, CoII, NiII, CuII, and ZnII). An uptake of 1 equiv of M...
52 citations
31 citations
TL;DR: The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn (2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.
Abstract: Bacteriophage lambda protein phosphatase (lambdaPP) 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. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP 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 lambdaPP. Escherichia coli cells overexpressing lambdaPP 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 lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.
27 citations
TL;DR: The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to thedinuclear center.
Abstract: Proton NMR spectra of FeIII-FeII recombinant single polypeptide human PAP (recHPAP) have been measured at, above, and below its pH optimum, as have the spectra of inhibited forms containing fluoride and phosphate, analogues of the substrates hydroxide and phosphate esters, respectively. The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to the dinuclear center. Thus, the group that is responsible for pKa,1 in the enzymatic activity versus pH profile functions as a "gatekeeper", whose protonation state controls anion binding to the mixed-valent dinuclear site. The correlation between the pKa values observed in kinetics studies and for the spectroscopic changes strongly suggests that this group is the nucleophilic hydroxide that attacks the phosphate ester substrate.
17 citations
01 Jan 2002
TL;DR: Results demonstrate that the differences in spectroscopic and kinetics properties previously reportedd for mammalian PAPs are the result of proteolytic cleavage, a potential physiological mechanism for regulating the activity of PAP in vivo.
Abstract: Abstract Recombinantt human purple acid phosphatase (recHPAP) provides a convenient experimentall system for assessing the relationship between molecular structure and enzymaticc activity in mammalian purple acid phosphatases (PAPs). RecHPAP is a single polypeptidee protein with properties similar to those of uteroferrin (Uf) and other PAPs isolatedd as single polypeptide chains, but its properties differ significantly from those of bovinee spleen PAP (BSPAP) and other PAPs isolated as proteolytically "cl ipped" forms. Incubationn of recHPAP with trypsin results in proteolytic cleavage in an exposed region nearr the active site. The product is a tightly associated two-subunit protein whose collectivee spectroscopic and kinetics properties resemble those of BSPAP. These results demonstratee that the differences in spectroscopic and kinetics properties previously reportedd for mammalian PAPs are the result of proteolytic cleavage. Masss spectrometry shows that a 3-residue segment, D-V-K, within the loop region is excisedd by trypsin. This finding suggests that interactions between residues in the excisedd loop and one or more of the groups that participate in catalysis are lost or alteredd upon proteolytic cleavage. Analysis of available structural data indicates that the mostt important such interaction is that between Asp l46 in the exposed loop and active sitee residues Asn91 and His92. Loss of this interaction should result in both an increase inn the Lewis acidity of the Fe ion and an increase in the nucleophilicity of the Feboundd hydroxide. Proteolytic cleavage thus could constitute a potential physiological mechanismm for regulating the activity of PAP in vivo.
2 citations