Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

Schenk, Gerhard; Gahan, Lawrence R.; Carrington, Lyle E.; Mitić, Nataša; Valizadeh, Mohsen; Hamilton, Susan; Jersey, John de; Guddat, L.W.

doi:10.1073/pnas.0407239102

Cited by 138 publications

(261 citation statements)

References 71 publications

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“…[10][11][12] Structural comparison to other enzymes indicates that GpdQ belongs to the group of R/ sandwich metallophosphoesterases, which also includes purple acid phosphatases (PAPs), the 3′-5′ cyclic nucleotide diesterase Rv0805 from Mycobacterium tuberculosis, and Mre11 nuclease. [13][14][15][16][17][18][19][20][21][22][23] The active site ( ion in the R site is coordinated by four amino acid residues, a terminal water ligand, and a hydr(oxide) molecule that bridges the two metal ions. In the site, the metal ion may also be coordinated by four amino acid residues but, based on spectroscopic and kinetic data, is predicted to be less tightly bound ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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Abstract:The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K d values for the metal ions in the R and sites to be 29 and 344 µM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the site increases substantially (K d ) 56 µM), while that of the R site is not greatly affected (K d ) 17 µM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k obs1 ), the assembly of a catalytically active binuclear center (k obs2 ), and subsequent slower structural rearrangements to optimize catalysis (k obs3 ). These three phases depend on the concentration of substrate ([S]), with k obs1 and k obs2 reaching maximum values at high [S] (354 and 38 s -1 , respectively), whereas k obs3 is reduced as [S] is increased. The k cat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s -1 ) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k obs3 is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate-and metal-ion-induced fit to optimize catalysis.

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Section: Introductionmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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“…In model (b) the bridging (hydr)oxo group acts as nucleophile. 5,10,12,13,14 The catalytic activity of a metal ion is largely governed by its Lewis acidity. The stronger the Lewis activity, the stronger is the effect of the metal ion on substrate activation.…”

Section: Introductionmentioning

confidence: 99%

Ga^III Complexes as Models for the M^III Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate

2010

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The effects of a series of Ga III complexes with tripodal ligands on the hydrolysis rate of the activated phosphate diester bis(2,4-dinitrophenyl)phosphate (BDNPP) have been investigated. In particular, the influence of the nature of the ligand donor sites on the reactivity of Ga III which represents a mimic of the Fe III ion in purple acid phosphatase has been evaluated. It has been shown that replacing neutral nitrogen donor atoms and carboxylate groups by phenolate groups enhanced the reactivity of the Ga complexes. Bell-shaped pH-rate profiles and the measured solvent deuterium isotope effects are indicative of a mechanism that involves nucleophilic attack on the coordinated substrate by Ga-OH. The trend in reactivity found for the different Ga complexes reveals that of the two effects of the metal, Lewis acid activation of the substrate and nucleophile activation, the latter one is more important in determining the intrinsic reactivity of the metal catalyst. The relevance of the present findings for the modulation of the activity of the M III ion in purple acid phosphatase whose active site contains a phenolate (tyrosine side chain) is discussed.

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“…In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.…”

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Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

et al. 2012

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Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K i values in the 30-60 lM range. Ligand efficiency values are 0.39-0.44 kcal ⁄ mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.Key words: X-ray, crystallography, drug design, fragment screening, purple acid phosphatase, osteoporosis Purple acid phosphatases (PAP) are metalloenzymes that hydrolyse phosphate esters and anhydrides, generally at low pH values (1). The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) (2,3). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II ⁄ III) in mammals (4,5) or a Zn(II) or Mn(II) in plants (6,7). Mammalian PAP is a 35 kDa monomeric protein also known as tartrate-resistant acid phosphatase (TRAP or TRAcP). In contrast, plant PAP is a 110 kDa homodimer, with each subunit consisting of two domains, an N-terminal one whose function is unknown and a catalytic C-terminal domain that strongly resembles the overall structure of the mammalian enzyme (1). Crystal structures of human (8), pig (9), rat (10,11) and plant PAPs (12,13) have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.A number of biological roles for PAP have been proposed. These include (i) the transport of iron from the mother to the developing foetus during gestation (14); (ii) bone resorption in osteoclasts (evidence for this activity is derived from the effect of PAP on osteoclasts that were cultured on cortical bone slices (15) and from t...

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Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

Cited by 138 publications

References 71 publications

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Ga^III Complexes as Models for the M^III Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate

Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

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Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

Cited by 138 publications

References 71 publications

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

GaIII Complexes as Models for the MIII Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate

Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

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Ga^III Complexes as Models for the M^III Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate