Crystal Structures of Recombinant Human Purple Acid Phosphatase With and Without an Inhibitory Conformation of the Repression Loop

Sträter, Norbert; Jasper, Beate; Scholte, M. E.; Krebs, Bernt; Duff, Anthony P.; Langley, David B.; Han, Runlin; Averill, Bruce A.; Freeman, H.C.; Guss, J. Mitchell

doi:10.1016/j.jmb.2005.04.014

Cited by 78 publications

(85 citation statements)

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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 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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“…[4][5][6] Mammalian PAPs are $35 kDa monomeric proteins with a redox active Fe(III)-Fe(II/III) centre in their active sites. 7,8 While it is the reduced form that is catalytically active, only the structures of the inactive diferric forms of PAPs from human (hPAP) 9 and pig (pPAP) 10 have been determined by X-ray crystallography. In contrast, most plant PAPs are 110 kDa homodimers with redox-inactive Fe(III)-Zn(II) or Fe(III)-Mn(II) centres.…”

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“…In contrast, hPAP can only be obtained in very small quantities, using a baculoviral recombinant expression system. 33 The validity of using rkbPAP and pPAP as models of the human enzyme is supported by a number of factors including (i) the high degree of structural conservation in their substrate-binding pockets, 9,10,[14][15][16] (ii) their conserved reaction mechanism(s), 1 and (iii) the similarity of inhibition constants for a range of inhibitors reported for several animal (including human) and plant PAPs (e.g., the fluoride inhibition constants of rkbPAP, pPAP and hPAP are 170, 120 and 170 lM, respectively). 1,11,17,[28][29][30][31] Furthermore, mammalian and rkbPAPs have similar catalytic efficiencies and catalyse the hydrolysis of similar substrates.…”

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confidence: 99%

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Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

McGeary

Vella

Mak

et al. 2009

Bioorganic & Medicinal Chemistry Letters

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a b s t r a c tPurple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The K i values of these compounds are as low as 4 lM, the lowest reported to date for a PAP inhibitor.

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Crystal Structures of Recombinant Human Purple Acid Phosphatase With and Without an Inhibitory Conformation of the Repression Loop

Cited by 78 publications

References 53 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

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

Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

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