Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Gene 377 (2006) 12 – 20 www.elsevier.com/locate/gene

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase J.U. Flanagan a,⁎, A.I. Cassady a , G. Schenk b , L.W. Guddat b , D.A. Hume a,c a

c

Cooperative Research Centre for Chronic Inflammatory Disease, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia b School of Molecular and Microbial Sciences, University of Queensland, Queensland, 4072, Australia Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Queensland, 4072, Australia Received 29 November 2005; received in revised form 21 February 2006; accepted 22 February 2006 Available online 5 April 2006 Received by C. Saccone

Abstract Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of ∼35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the ∼55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. © 2006 Elsevier B.V. All rights reserved. Keywords: Tartrate-resistant; Metalloenzyme; Structure; Fenton catalyst

1. Introduction Purple acid phosphatases (PAPs; the mammalian enzymes from human, mouse and rat are better known as type 5 acid phosphatases, herein referred to as ACP5) belong to a diverse group of binuclear metallohydrolases that have been identified and characterized in plants, animals and fungi (Klabunde and Krebs, 1997; Schenk et al., 2000a,b). PAP-like sequences have also been identified in a small number of bacteria (Schenk et al., 2000a). PAPs contain a binuclear metal centre of the form Fe(III)–M(II), where M =Fe, Zn or Mn. Their characteristic purple colour is due to a tyrosine to Fe(III) charge transfer transition (Antanaitis et al., 1983). To date, the mammalian enzymes extracted from human, pig, cow, Abbreviations: PAP, Purple acid phosphatase; ACP5, tartrate-resistant ∼ 35 kDa acid phosphatase 5; PAPL, Purple acid phosphatase long form; Hsa, Homo sapiens; INDELS, insertions and deletions; rmsd, root-mean-squaredeviation. ⁎ Corresponding author. Tel.: +61 7 33462075; fax: +61 7 33462101. E-mail address: [email protected] (J.U. Flanagan). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2006.02.031

rat and mouse are all monomers of ∼35 kDa, contain a redoxactive Fe(III)–Fe(II/III) centre, and are distinguished from other acid phosphatases because of their resistance to inhibition by L(+) tartrate (Oddie et al., 2000). The plant PAPs that have been characterized are larger and contain either two identical subunits of ∼55 kDa (Beck et al., 1986; Schenk et al., 1999), or two dissimilar subunits of 63 and 57 kDa (Bozzo et al., 2002). Notably, the plant enzymes have been isolated with Fe(III)–Zn(II) (Beck et al., 1986; Schenk et al., 1999; Durmus et al., 1999a) and Fe(III)–Mn(II) (Schenk et al., 1999, 2001) catalytic centres. The three-dimensional structures of red kidney bean (Klabunde et al., 1996), pig (Guddat et al., 1999), rat (Lindqvist et al., 1999; Uppenberg et al., 1999), sweet potato (Schenk et al., 2005) and human (Sträter et al., 2005) PAPs show that there is a high degree of conservation among the residues around the catalytic centre. There are seven invariant amino acids that coordinate the metal ions, and map to the loops at the end of the β-strands that form the core of the enzyme. Five sequence motifs created from conserved blocks of residues have previously been used to query genome databases to

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search for PAP genes (Schenk et al., 2000a,b). Multiple PAP-like isoforms have been identified in the genomes of Arabidopsis thaliana (Schenk et al., 2000b), sweet potato (Schenk et al., 1999; Durmus et al., 1999b), tomato (Bozzo et al., 2002), soybean (Schenk et al., 2000b), red kidney bean (Schenk et al., 2000b) and potato (Zimmermann et al., 2004). Of particular interest was the observation that plants appear to contain at least three types of PAPs, a dimeric form consisting of two identical polypeptides of ∼55 kDa, a dimeric form consisting of two different subunits, and an as of yet uncharacterized ∼35 kDa form homologous to mammalian enzymes (Schenk et al., 2000b; Bozzo et al., 2002). In contrast, only a single low molecular weight isoform has been detected in mammalian genomes (Grimes et al., 1993; Ling and Roberts, 1993; Leach et al., 1994). No PAPs have yet been reported from Drosophila or other insect genomes (Oddie et al., 2000). Although the precise physiological role(s) of ACP5 remains uncertain its high expression levels in both osteoclasts and (to a lesser extent) in macrophages have led to the suggestion that ACP5 may be involved in bone degradation (Hayman et al., 1996) and bacterial killing (Raisänen et al., 2005). ACP5 catalyses the hydrolysis of a range of phosphate esters and anhydrides, including 2umbelliferylphosphate, p-nitrophenolphosphate, phosphotyrosine analogues and pyrophosphate (Nash et al., 1993; Hayman et al., 1989; Valizadeh et al., 2004). Other physiologically more relevant substrates include ATP (Mitić et al., 2005) and the bone matrix phosphoproteins, osteopontin and bone sialoprotein. Dephosphorylation of these bone matrix proteins reduces the ability of osteoclasts to attach to the bone surface (Ek-Rylander et al., 1994). In addition to its phosphatase activity, the reduced Fe(III)–Fe(II) enzyme can generate hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction (Sibille et al., 1987; Oddie et al., 2000; Halleen et al., 2003). These reactive oxygen species (ROS) can degrade type I collagen (Halleen et al., 1999), and ROS production in activated macrophages isolated from ACP5 overexpressing transgenic mice is also associated with increased microbial killing (Raisänen et al., 2005). Due to its crucial role in bone metabolism, ACP5 is a target for the development of antiosteoporotic drugs (Valizadeh et al., 2004). ACP5 activity in bone metabolism is partly compensated by the activity of the structurally unrelated lysosomal acid phosphatase (LAP). A double LAP/ACP5 knockout mouse has greater penetrance of skeletal abnormalities than either of the single gene knockouts alone (Saftig et al., 1997). It may be possible that other PAP isoforms yet to be identified can act as Fenton catalysts and/or non-specific phosphatases, and thus also have a compensatory effect in the absence of ACP5. In the current study we describe the identification, and molecular modeling of a previously unknown human gene product that is likely to encode a PAP-like isoform larger than, and genetically distinct to, the previously characterized ACP5 enzyme. 2. Methods 2.1. Identification of new acid phosphatase isoforms Homologs of human ACP5 (accession number NP_001602) were sought using a PSI-BLAST search (Altschul and Koonin,

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1998) of the NCBI non-redundant database (Oct-19-2004: 2,095,794 sequences). The search was performed using default parameters and the BLOSUM62 matrix. Only sequences below the E-value threshold of 0.005 were included in the generation of the position specific score matrix (PSSM). All sequences were extracted and trimmed to the region identified as homologous to human ACP5. 2.2. Comparative modeling of the novel human PAP isoform A template structure for modeling was identified from the PSI-BLAST search described above. Alignments between the target and template sequences were generated using two approaches. Firstly, the pairwise alignment generated during the PSI-BLAST search was kept without further alteration. Secondly, all matches occurring between the new human PAP-like sequence and the closest protein databank (pdb) entry (4KBP, red kidney bean PAP) were extracted, trimmed to the homologous region as defined in the PSI-BLAST output, and aligned using CLUSTALX. Consequently, this alignment was used for phylogenetic analyses. 2.3. Secondary structure analysis Secondary structure elements were predicted using JPRED (Cuff and Barton, 2000), PSIPRED (McGuffin et al., 2000) and PHD (through the PREDICTPROTEIN metaserver) (Rost, 1996), while the secondary structure for the red kidney bean PAP template was taken from the pdb entry 4KBP. The fine adjustment of gap positions was assessed by superimposition of predicted and observed secondary structures. 2.4. Model construction Construction of an atomic model for the novel human PAP was performed using MODELLER v6.0 as implemented in the HOMOLOGY module of the INSIGHT software suite (Accelrys). The red kidney bean PAP structure, 4KPB, was used as the primary template. Fragments from the pig (1UTE) and sweet potato (1XZW) PAP structures were also used in the construction of the loops connecting β13 and α4, and, β14 and α5, respectively. All other loops were generated automatically using MODELLER. Structure refinements were performed using simulated annealing and molecular dynamics as implemented in the highest refinement level of MODELLER package. An ensemble of 15 models was calculated and the structures assessed for stereochemical correctness using PROCHECK (Laskowski et al., 1993). A composite model was constructed from the ensemble of substructures through the combination of regions displaying good stereochemistry. Manual adjustment of sidechain rotamers was carried out to alleviate any steric clashes. These coordinates were used for subsequent rounds of model building. The root-mean-square-deviation (rmsd) between the template and modeled structures was assessed across all matching Cα atoms.

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3. Results and discussion 3.1. Sequence similarity searches In order to identify remote homologs of the known mammalian PAPs an iterative PSI-BLAST search of the non-redundant database was performed using the human ACP5 sequence (accession number NP_001602). The search was stopped once sequences with known biological function different from the PAPs were identified. All matches extracted by the search had Evalues of 8e− 16 or less indicating they are extremely unlikely to have occurred by chance. All known mammalian PAP sequences were present in the top ten matches (human, mouse (Marshall et al., 1997), rat (Ek-Rylander et al., 1997) and pig (Wynne et al., 1995)), and have amino acid sequence identities of N83%. PAP sequences were also found in frog (Xenopus laevis), fish (Danio rerio; Tetraodon nigroviridis) and two species of nematodes (Caenorhabditis briggsae; Caenorhabditis elegans). Identities range from ∼30% to 60%. The distantly related, red kidney bean PAP (4KBP) was within the top 100 matches with a sequence identity of 13%. Sequences more distantly related to the query were also identified in mammalian, bird, fish, insect and nematode genomes (Table 1). This table also indicates the number of new genes found in each species. (The new plant-like sequences are denoted PAPL to represent the long form of PAP.) The effectiveness of the position specific score matrix (PSSM) generated by PSI-BLAST in covering the PAP-like sequence space was assessed by analysis of A. thaliana sequences. It has been reported previously that this genome is likely to contain up to 29 PAP-like sequences (Li et al., 2002). Our search retrieved all previously detected candidates, indicating that the PSSM generated by the iterative PSI-BLASTsearch is sufficient for the identification of all distantly related PAP homologs. The significant outcome of this search is that plants are not the only organisms with multiple isoforms of PAP-like genes. The discovery of a human gene whose

product has 25% identity with red kidney bean PAP (Table 2) demonstrates for the first time that mammalian organisms may also possess at least two PAPs with differing molecular weight, i.e. a ∼35 kDa (ACP5) and a ∼55 kDa form. To identify other mammalian relatives of the new PAP-like genes, each animal sequence listed in Table 2 was used as a query in further PSI-BLASTsearches but no further novel sequences were identified. 3.2. Phylogenetic analysis The relationship between various animal and plant PAP and PAPL sequences was further investigated through phylogenetic analysis (Fig. 1). In agreement with earlier studies (Schenk et al., 2000b; Zimmermann et al., 2004) a distinct partition between the previously characterized animal and plant enzymes is apparent. The novel animal PAPL sequences are indeed more closely related to the known plant isoforms. 3.3. Secondary structure prediction and sequence analysis Red kidney bean PAP (pdb entry 4KBP), the closest sequence homolog to human PAPL (Hsa_PAPL), was used as a template for the generation of a structural model. Alignments between the target and template sequences were taken from the PSI-BLAST output (vide supra; Fig. 2). For comparison, the human ACP5 sequence was also superimposed to illustrate the dissimilarity between the two human isoforms. The alignment quality between the red kidney bean PAP and Hsa_PAPL was assessed through the superposition of secondary structure elements. The predicted secondary structure elements of Hsa_PAPL align well with those from red kidney bean PAP even though the sequence identity is only ∼25% and the overall sequence similarity is 37%. A number of insertions or deletions (INDELs) occur between the target and template sequences outside the regions of defined secondary structure, and correspond to loop regions in the plant enzyme. Of

Table 1 Sequence identity between the putative high molecular weight animal, a red kidney bean and the human low molecular weight isoforms of purple acid phosphatases

Hsa_ACP5 Hsa_PAPL1 Mmu_PAPL1 Dme_PAPL1 Dme_PAPL2 Dme_PAPL3 Ame_PAPL1 Aga_PAPL1 Tni_PAPL1 Cel_PAPL1 Cel_PAPL2 Cel_PAPL3 Cel_PAPL4 Cbr_PAPL1 Cbr_PAPL2 Pvu_PAP

Hsa ACP5

Hsa Mmu PAPL1 PAPL1

Dme PAPL1

Dme PAPL2

Dme PAPL3

Ame Aga Tni Cel Cel Cel Cel Cbr Cbr Pvu PAPL1 PAPL1 PAPL1 PAPL1 PAPL2 PAPL3 PAPL4 PAPL1 PAPL2 PAP

100

15 100

14 52 52 100

14 56 54 65 100

16 53 51 62 64 100

13 54 51 60 59 59 100

16 86 100

13 53 54 69 67 64 66 100

15 51 49 46 62 58 50 50 100

14 51 50 48 47 49 48 48 57 100

14 45 44 48 47 49 48 48 57 92 100

15 36 33 36 33 36 34 32 37 40 32 100

17 38 38 40 39 39 37 39 35 34 36 35 100

16 35 35 35 34 36 36 34 37 34 34 80 32 100

16 36 33 37 36 36 35 36 38 34 34 65 34 66 100

16 25 26 26 26 25 26 24 27 27 27 24 28 26 25 100

Species identifiers: Hsa, Homo sapiens; Mmu, Mus musculus; Dme, Drosophila melanogaster; Ame, Apis mellifera; Aga, Anopheles gambiae; Tni, Tetraodon nigroviridis; Cel, Caenorhabditis elegans; Cbr, Caenorhabditis briggsae; Pvu, Phaseolus vulgaris, red kidney bean. PAPL, purple acid phosphatase long form. Sequence identities are as calculated by BLAST.

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Table 2 Sequence characteristics of the newly identified PAP isoforms Source HMW plant-like PAPs Mammals Insects

Fish Nematodes

Structural relatives

LMW PAPs Mammalian Fish

Nematodes Frogs

Accession number

Identifier

Length

1

2

3

4

5

NP_001004318 NP_780528 NP_727465 AAM49866 NP_727464 XP_396873 XP_321039 CAG10668 NP_506864 T21181 NP_502892 NP_506191 CAE57394 CAE57393 4KBP 1UTE 1QHW

Hsa_PAPL1 Mmu_PAPL1 Dme_PAPL1 Dme_PAPL2 Dme_PAPL3 Ame_PAPL1 Aga_PAPL1 Tni_PAPL1 Cel_PAPL1 Cel_PAPL2 Cel_PAPL3 Cel_PAPL4 Cbr_PAPL1 Cbr_PAPL2 Pvu_PAP Ssc_PAP Rno_ACP5

438 496 458 450 453 451 472 378 431 424 438 455 416 416 459 340 327

GDLG GDMG GDMG GDMG GDMG GDMG GDMG – GDLG GDLG GDLS GDLG GDLS GDLS GDLG GDWG GDWG

GDFAY GDFAY GDFAY GDFAY GDFAY GDFAY GDFAY –DFAY GDFAY GDFAY GDLAY GDIAY GDLAY GDIAY GDLSY GDNFY GDNFY

GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHE GNHD GNHD

TMGH TMGH LYGH TYGH TYGH TFGH TYGH TMGH TMGH ADQ VMFH TFQH VMFH VMFH VLMH VAGH VAGH

AHEH AHEH AHEH AHEH AHEH AHEH AHEH AHEH AHEH AHEH GHKH GHEH GHKH GHKH GHVH GHDH GHDH

NP_001602 AAH19160 CAG03307 NP_001002452 NP_999938 NP_492283 CAE67273 AAH81357 AAH73550 AAH72062

Hsa_ACP5 Mmu_ACP5 Tni_PAP Dre_PAP1 Dre_PAP2 Cel_PAP Cbr_PAP Xtr_PAP Xla_PAP1 Xla_PAP2

325 327 331 327 339 419 384 326 325 326

GDWG GDWG GDWG GDWG GDWG GDTG GDTG GDWG GDWG GDWG

GDNFY GDNFY GDNFY GDNFY GDNFY GDNIY GDNIY GDNFY GDNFY GDNFY

GNHD GNHD GNHD GNHD GNHD GNHD GNHD GNHD GNHD GNHD

VAGH VAGH VAGH VAGH VAGH ISGH VSGH VAGH VAGH VAGH

GHDH GHDH GHDH GHDH GHDH GHDH GHDH GHEH GHEH GHEH

Species identifiers: Hsa, Homo sapiens; Mmu, Mus musculus; Dme, Drosophila melanogaster; Ame, Apis mellifera; Aga, Anopheles gambiae; Tni, Tetraodon nigroviridis; Cel, Caenorhabditis elegans; Cbr, Caenorhabditis briggsae; Pvu, Phaseolus vulgaris, red kidney bean; Ssc, Sus scrofa; Rno, Rattus norvegicus; Dre, Danio rerio; Xtr, Xenopus tropicalis; Xla, Xenopus laevis. PAPL, purple acid phosphatase long form. The tyrosine residue highlighted in the second cluster of conserved residues, is proposed to be involved in the formation of the charge transfer transition that is responsible for the purple colour of the PAPs, indicating that the novel sequences identified in this study are PAP-like. HMW, high molecular weight; LMW, Low molecular weight.

these, an eight-residue deletion occurs in the PAPL sequence between α3 and α4, and one of four residues between α4 and β11, while insertions of six and nine residues occur in the loops between β12 and α5, and β13 and α6, respectively. The metal-ligating residues are distributed over five clusters, all of which are conserved in Hsa_PAPL (Table 2). These are located at the C-terminal ends of β8, β9, β13, β14 and the Nterminal end of α3 (Fig. 2). 3.4. Active site structure The analysis of the sequence alignment in Fig. 2 and the homology model in Fig. 3 illustrates that the active site of Hsa_PAPL is similar to that of other PAPs, allowing for the coordination of two metal ions by seven conserved amino acids. The metal ion composition of the novel human PAP is unknown, but due to its homology to other PAPs it is likely that Fe(III) is one of the metal ions, enabling the formation of the characteristic tyrosinate to Fe(III) charge transfer complex (see above). In known PAPs the second metal ion is either Fe(II), Zn(II) or Mn (II), and it is anticipated that either of these metal ions may be present in Hsa_PAPL. In addition to the metal ions, a phosphate

group was modeled into the active site in a metal ion-bridging mode (Fig. 3). A similar coordination mode has been observed in red kidney bean PAP (Klabunde et al., 1996). Both metal ions are thus coordinated by four conserved amino acids and an oxygen atom from the phosphate. The remaining coordination position on each metal ion is likely to be occupied by a bridging hydroxide ligand, as observed in the crystal structure of pig PAP (Guddat et al., 1999) and hypothesized in the red kidney bean PAP structure (Sträter et al., 1995). The metal-coordinating residues are listed for Hsa_PAPL, Hsa_PAP and red kidney bean PAP in Table 3, and the spatial arrangement of these residues in the Hsa_PAPL model is illustrated in Fig. 3. Both the template structure and the homology model are very similar as judged by an overall rmsd of 1.13 Å for their respective Cα atoms. However, in contrast to the conserved catalytic centre changes in the length of the α3/α4, β12/α5 and β13/α6 loops between the target and template suggest substantial differences in the structure of the active site groove outside of the catalytic centre (Fig. 4). The α3/α4 deletion reduces the height of the active site on one face, while the β12/α5 insertion increases the size of a surface loop at the entrance to the active site groove. The true position of the β13/α6 loop, its conformation, and thus its effect

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Fig. 1. Phylogenetic tree showing the relationship between the known and putative purple acid phosphatases listed in Table 1. Bootstrap numbers (1000 replicates) are indicated at internal nodes. The scale bar indicates the number of substitutions per site. Explanation of the codes is given in Table 1.

on the active site are difficult to determine. In the template structure, the C-terminus of β13 and the β13/α6 loop region packs against a loop defined by residues 346 to 357 (Hsa_PAPL numbering). Sequence differences between the two enzymes in this region indicate structural changes in Hsa_PAPL to accommodate the insertion shown in Fig. 2. The loop was modeled in three conformations, (1) using the boundary of α6 observed in the template structure; (2) using the consensus secondary structure prediction for α6; and (3) using the PSIPRED results, where α6 is predicted to be eight residues longer than the consensus prediction. The model based on the PSIPRED prediction is preferred over the other two combinations, as it reduces the size of the unstructured

loop through extension of the helix present in the template. In the current model the β13/α6 insertion increases the height of an active site face. In summary, these structural differences between Hsa_PAPL and red kidney bean PAP in the vicinity of the active site are expected to lead to alterations in their respective substrate specificities, suggesting alternative biological roles. 3.5. Phosphate coordination Residues predicted to be involved in phosphate coordination in Hsa_PAPL, red kidney bean and sweet potato PAPs are listed in Table 3. Hsa_PAPL residues Asn175, His176, His303 and

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Fig. 2. Alignment of purple acid phosphatase and purple acid phosphatase-like sequences from human and red kidney bean. The PSI-BLAST alignment of human ACP5 and Hsa_PAPL was superimposed onto that of the red kidney bean and Hsa_PAPL. The secondary structure for red kidney bean is in black, and was taken from the description in the pdb file 4KBP; a consensus prediction is shown for the purple phosphatase-like isoform and is based on the JPRED, PSIPRED and PREDICTPROTEIN methods (green). Secondary structure elements are numbered according to 4KBP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

His305 are conserved across all three isoforms, and are thus expected to have similar mechanistic roles. A Hsa_PAPL residue equivalent to His295 or His296 in red kidney bean PAP is not unambiguously identified in the current model due to the insertion in the β13/α6 loop (see Section 3.4). Moreover, the sweet potato PAP residue Glu365, which has been proposed to act as proton donor to the substrate leaving group at low pH (Schenk et al., 2005), does not have an equivalent residue in the red kidney bean enzyme, but does align with Glu345 in Hsa_PAPL. However, the current Hsa_PAPL model cannot verify that Glu345 is spatially equivalent to Glu365 in sweet potato PAP.

(Fig. 2). The rmsd for the two structures is 1.5 Å for 194 Cα atoms. The most striking difference between the two human isoforms is the presence of an N-terminal β-sheet domain in Hsa_PAPL (Figs. 2 and 5). This domain has only been observed in the 55 kDa isoforms identified in plants and has as of yet no known function. Additional differences exist in the positions of loops between the human ACP5, Hsa_PAPL and red kidney bean PAP. The α5/

3.6. Dimer interface Red kidney bean PAP is a homodimer with the two subunits linked via a disulfide bridge formed by residue Cys345 from each subunit (Klabunde et al., 1996). The possibility that Hsa_PAPL may form a dimer in a similar manner was assessed by superimposing two Hsa_PAPL subunits onto the dimeric red kidney bean enzyme. An equivalent Cys residue is not found in the human enzyme, nor is there any other Cys residue in this region that could form a disulfide bond. Variations in INDELs in the region around Cys345 in the template sequence and the equivalent region in Hsa_PAPL (Fig. 2) further indicate that the latter is not likely to form a dimer. 3.7. Comparison to the human ACP5 structure The catalytic centres in Hsa_PAPL and the recently determined structure of human ACP5 (Sträter et al., 2005) are very similar, with the seven metal-coordinating residues conserved with respect to identity and relative position in the alignment

Fig. 3. Structure of the modeled catalytic site of the new human purple acid phosphatase (Hsa_PAPL). The interaction between the tyrosine and the Fe(III) responsible for the purple colour of the purple acid phosphatases is illustrated by a green dashed line. A phosphate has been modeled based on that observed in the red kidney bean structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 3 Residues involved in metal and phosphate coordination in Hsa_PAPL, red kidney (Pvu) and sweet potato (Iba) PAP isoforms

Fe(III)

Me(II)

Phosphate

Hsa_PAPL

Pvu_PAPL

Iba_PAPL

Asp111 Asp140 Tyr143 His305 Asp140 Asn175 His256 His303 Asn175 His176 His303 His305 – – Glu345

Asp135 Asp164 Tyr167 His325 Asp164 Asn201 His286 His323 Asn201 His202 His323 His325 His295 His296 –

Asp134 Asp163 Tyr166 His324 Asp163 Asn200 His285 His322 Asn200 His201 His322 His324 – His295 Glu365

Species identifiers: Hsa, Homo sapiens; Pvu, Phaseolus vulgaris, red kidney bean; Iba, Ipomoea batatas, sweet potato.

β12 loop in ACP5 (Fig. 2) is larger than that in Hsa_PAPL or the red kidney bean enzyme, and has been identified as a repression loop (Uppenberg et al., 1999). Upon proteolytic cleavage within this loop the reactivity of ACP5 can increase significantly and the substrate specificity can alter (Ek-Rylander et al., 1997; Ljusberg et al., 1999; Mitić et al., 2005). Specifically, proteolysis in the repression loop of human ACP5 turns this enzyme into a potent ATPase (Mitić et al., 2005). In contrast, the corresponding region in Hsa_PAPL is not expected to be proteolytically cleaved as assessed using the MEROPS database (http://merops.sanger.ac. uk), and thus its catalytic activity and substrate specificity are unlikely to be modified in a manner similar to those of ACP5. An essential part of the catalytic mechanism requires the protonation of the leaving alcohol group (Schenk et al., 2005). For human ACP5 His90 (corresponds to His202 and His201 in red kidney bean and sweet potato PAP, respectively; Table 3) is the likely proton donor (Kaija et al., 2002; Funhoff et al., 2005). In Hsa_PAPL, His176 is in a position equivalent to His90 in

Fig. 4. Comparison of the differences between the predicted structure of the new human purple acid phosphatase-like (green) and the template structure of red kidney bean purple acid phosphatase (4KBP; red). The grey surface represents the common core of the two structures, while the regions of insertions and deletions are represented as ribbons. The two metal atoms of the catalytic centre are illustrated as blue spheres.

Fig. 5. Comparison of the two human purple acid phosphatase isoforms. The grey surface represents the common core structure between the two isoforms. Hsa_PAPL is represented as green ribbon whereas the recently determined Hsa_PAP (pdb entry 1WAR) is in red. The N-terminal β-sheet domain previously associated with the larger plant isoforms is illustrated in the Hsa_PAPL structure. The differences in loop sizes for the β12/α5 and β13/α6 are shown. The position of the active site is indicated by an arrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ACP5 both in sequence and spatially, suggesting that both enzymes utilize a similar mechanistic strategy. 4. Conclusion Based on the observation that multiple PAPs exist in plant genomes (Schenk et al., 2000b), we explored the possibility that the mammalian genomes also contain more than one PAP-like gene. A PSI-BLAST search using the human PAP sequence as a query identified the presence of at least one other PAP-like transcript encoding a protein distinct from known ACP5s (∼36 kDa) in mammalian, insect and nematode genomes. The new isoform, designated PAPL, is larger than ACP5s (calculated molecular weight: ∼55 kDa), and more closely related to the high molecular weight PAPs characterized from red kidney beans (Beck et al., 1986; Klabunde et al., 1996) and sweet potatoes (Schenk et al., 1999, 2001, 2005; Durmus et al., 1999a). These results establish the presence of distinct genes encoding both low and high molecular weight PAPs in animal genomes. Modeling studies illustrate that the predicted fold for Hsa_PAPL is consistent with that observed for red kidney bean (Klabunde et al., 1996) and sweet potato (Schenk et al., 2005) PAPs. The seven invariant amino acid residues which coordinate the two metal ions in the active site of PAPs are conserved in Hsa_PAPL. This includes a tyrosine (143) that coordinates to Fe(III) in the active site of known PAPs, providing the molecular basis for the characteristic purple colour of these enzymes. Apart from the metal ion-coordinating amino acids other residues implicated in various aspects of the catalytic mechanism are conserved in Hsa_PAPL. Specifically, the proton donor to the leaving group (His176), and possibly the reaction-initiating nucleophile (an Fe(III)-bound hydroxide group) are present in Hsa_PAPL, indicating that the novel human PAP employs a catalytic mechanism similar to that proposed for other PAPs (Schenk et al., 2005). While the first coordination sphere is highly conserved amongst PAPs (including Hsa_PAPL) variations in loops in the

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vicinity of the active site are likely to affect their respective substrate specificities. Consequently, depending on the nature of their preferred substrates the biological roles of various PAPs may differ. The precise physiological function for PAPs is not known but while ACP5s from human, mouse and rat are associated with bone metabolism (Hayman et al., 1996; Oddie et al., 2000), plant PAPs are believed to be involved in phosphate acquisition (Duff et al., 1994). Hsa_PAPL resembles plant PAPs in overall structure, but its substrate specificity is likely to be different from both plant PAPs and ACP5s (see above). Consequently, while the physiological function of Hsa_PAPL currently remains obscure it is likely to fulfill a role different from other PAPs. Since ACP5 has become a target for the development of anti-osteoporotic drugs (Valizadeh et al., 2004) it will be essential (i) to establish the role of Hsa_PAPL, and to (ii) assess the biological effect of its inhibition by the abovementioned drugs. Acknowledgements The authors thank the Computational Chemistry and Biology Unit of the ARC Special Research Centre for Functional and Applied Genomics. This work was supported in part by the Cooperative Research Center for Chronic Inflammatory Disease, the National Health and Medical Research Council of Australia and the Australian Research Council (Grant DP558652). JUF is a NHMRC Howard Florey Centenary Fellow. The assistance of Ms Eleanor Leung in the preparation of this manuscript is gratefully appreciated. References Altschul, S.F., Koonin, E.V., 1998. Iterated profile searches with PSI-BLAST: a tool for discovery in protein databases. Trends Biochem. Sci. 23, 444–447. Antanaitis, B.C., Aisen, P., Lilienthal, H.R., 1983. Physical characterization of twoiron uteroferrin: evidence for a spin-coupled binuclear iron cluster. J. Biol. Chem. 258, 3166–3172. Beck, J.L., McConaghie, L.A., Summors, A.C., Arnold, W.N., de Jersey, J., Zerner, B., 1986. Properties of a purple acid phosphatase from red kidney bean: a zinc–iron metalloenzyme. Biochim. Biophys. Acta 869, 61–68. Bozzo, G.G., Raghothama, K.G., Plaxton, W.C., 2002. Purification and characterization of two secreted purple acid phosphatase isozymes from phosphatestarved tomato (Lycopersicon esculentum) cell cultures. Eur. J. Biochem. 269, 6278–6286. Cuff, J.A., Barton, G.J., 2000. Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40, 502–511. Duff, S.M., Sarath, G., Plaxton, W.C., 1994. The role of acid phosphatases in plant phosphorus metabolism. Physiol. Plant 90, 791–800. Durmus, A., et al., 1999a. The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas). Eur. J. Biochem. 260, 709–716. Durmus, A., Eicken, C., Spener, F., Krebs, B., 1999b. Cloning and comparative modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas). Biochim. Biophys. Acta 1434, 202–209. Ek-Rylander, B., Flores, M., Wendel, M., Heinegard, D., Andersson, G., 1994. Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. J. Biol. Chem. 269, 14853–14856. Ek-Rylander, B., Barkhem, T., Ljusberg, J., Ohman, L., Andersson, K.K., Andersson, G., 1997. Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase. Biochem. J. 321, 305–311.

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