Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase.
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Journal: 2005/February - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K(m)) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-A resolution (final R(free) value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to lambda protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.
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Proc Natl Acad Sci U S A 102(2): 273-278
PMID: 15625111

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

Departments of Biochemistry and Molecular Biology and Chemistry, School of Molecular and Microbial Sciences, University of Queensland, Brisbane 4072, Australia
To whom correspondence may be addressed. E-mail: ua.ude.qu@knehcs or ua.ude.qu.xobliam@taddug.ekul.
Edited by Brian W. Matthews, University of Oregon, Eugene, OR
Edited by Brian W. Matthews, University of Oregon, Eugene, OR
Received 2004 Oct 4; Accepted 2004 Nov 19.
Copyright © 2005, The National Academy of Sciences

Abstract

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)–Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a μ-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (kcat/Km) at pH 4.5, whereas its catalytic rate constant (kcat) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pKa of the leaving group. The crystal structure of the phosphate-bound Fe(III)–Mn(II) PAP has been determined to 2.5-Å resolution (final Rfree value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to λ protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Keywords: binuclear metal center, phosphate coordination
Abstract

Purple acid phosphatases (PAPs) belong to a diverse group of binuclear metallohydrolases that use metal ion centers to catalyze the hydrolysis of amides and esters of carboxylic and phosphoric acids (1, 2). PAPs have been observed in a wide range of animals, plants, and fungi. Genome database searches imply that a limited number of bacteria also possess PAP or PAP-like genes (3). In mammals, biological roles for PAPs have been suggested in iron transport (4), bone resorption by oste-oclasts (5, 6), dephosphorylation of erythrocyte phosphoproteins (7), and the production of hydroxyradicals and reactive oxygen species (8, 9). In plants, a role in the release of phosphate from organophosphates has been proposed for PAPs from Arabidopsis thaliana and tomato (11, §). Furthermore, these enzymes also have been shown to have alkaline peroxidase activity, implying a role for plant PAPs in defense against pathogen infection (11–13).

The active site of PAPs consists of a center with seven invariant amino acid residues coordinating to the two metal ions (14, 15). The characteristic purple color associated with these enzymes is attributed to a charge transfer transition from a tyrosine ligand to the Fe(III) in the binuclear center (16–18). In active PAPs, the second metal ion is always divalent, but its identity depends on the source of the enzyme. For example, in red kidney bean and soybean Fe(III)–Zn(II) PAPs have been isolated (19, 20), whereas all active mammalian PAPs characterized to date have Fe(III)–Fe(II) centers. Oxidation to the diferric form reversibly inactivates the mammalian enzymes, leading to the suggestion that PAP may be regulated by changes in redox potential within animal cells (21).

The three-dimensional structure of red kidney bean PAP showed that it is a 110-kDa homodimer, each subunit consisting of an N-terminal (residues 1–120) and a C-terminal (residues 121–432) domain. The latter contains the catalytic site, including the binuclear Fe(III)–Zn(II) center (22). The structures of the enzyme–phosphate and enzyme–tungstate complexes also have been reported (23). More recently, the crystal structures of pig PAP complexed with phosphate to 1.55-Å resolution (24) and two crystal forms of rat PAP have been determined to 2.2 and 2.8-Å resolution (25, 26). The mammalian enzymes are smaller monomeric proteins of ≈35 kDa, but their overall structure resembles that of the C-terminal domain observed in the red kidney bean enzyme. In plants, a second type of PAP has been identified that has a molecular weight and amino acid sequence closely related to those of the mammalian enzymes (15). The physicochemical properties of this form, including its metal ion content, are yet to be determined.

In sweet potato, both low and high molecular weight types of PAPs have been cloned and sequenced (15, 20, 27). Two isoforms of the high molecular mass (≈110 kDa) protein have been isolated to date. One of these contains an Fe(III)–Zn(II) center with kinetic and spectroscopic properties similar to those of red kidney bean PAP (27). In contrast, the other isoform contains a strongly antiferromagnetically coupled Fe(III)–Mn(II) center, the first of its kind in any biological system (20, 28). Compared with other PAPs, this isoform from sweet potato is further distinguished by its increased catalytic efficiency toward a broad range of both activated and unactivated phosphate esters (20) and its strict requirement for manganese (28). In contrast, in other PAPs the native divalent metal ion can be replaced by other divalent metal ions without major loss of activity (29–31).

The mechanism proposed for PAP-catalyzed reactions generally involves a nucleophilic attack of a metal-bound hydroxide on the phosphorus atom of the substrate, leading to hydrolytic cleavage of the phosphate-ester bond and the release of the leaving group (32). The precise identity of the nucleophile has been the subject of extensive debate. Measurements of the pH dependence of kinetic parameters of pig PAP have led to the proposition that a terminal, Fe(III)-bound hydroxide acts as the nucleophile during catalysis (32, 33). More recently, metal ion replacement studies using bovine PAP (31, 34) and spectroscopic characterization of pig PAP by using x-ray absorption (35) and electron-nuclear double resonance (36) support a model where the hydroxide bridging the two metal ions is the attacking nucleophile.

Spectroscopic and multifield saturation magnetization data imply that the manganese-dependent isoform of sweet potato PAP contains a μ-oxo instead of a μ-hydroxo bridge at pH 4.90 (28). Thus, if the attacking nucleophile in this enzyme at this pH is the bridging ligand, it would be the μ-oxo species rather than a μ-hydroxo species. Here, the pH dependence of kinetic parameters and the inhibition by inorganic phosphate of the Fe–Mn sweet potato enzyme are compared with other PAPs. Furthermore, the dependence of the catalytic rate on the leaving group pKa is assessed in combination with the three-dimensional crystal structure of the phosphate-bound complex. These data allow us to discuss mechanistic features that appear at present to be unique to this PAP. In the process of structure determination an unusual coordination mode for phosphate has been observed, reminiscent of the phosphate complex of the binuclear nickel enzyme urease from Bacillus pasteurii (37).

When measuring the pH dependence of the kinetic parameters, 4-nitrophenol phosphate was used as a substrate. n.d., not determined.

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Acknowledgments

We thank Dr. Harry Tong for assistance at beamline 14BM-C (Advanced Photon Source, Argonne National Laboratory). The use of the BioCARS Sector (Consortium for Advanced Radiation Sources) was supported by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program. Use of the BioCARS Sector 14 also was supported by National Institutes of Health/National Center for Research Resources Grant RR07707. Use of the Advanced Photon Source was supported by U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research Contract W-31-109-Eng-38.

Acknowledgments

Notes

Author contributions: G.S., L.R.G., S.E.H., J.d.J., and L.W.G. designed research; G.S., L.E.C., N.M., M.V., and L.W.G. performed research; G.S., L.R.G., N.M., and L.W.G. analyzed data; and G.S., L.R.G., and L.W.G. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PAP, purple acid phosphatase; PP, protein phosphatase.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1XZW).

Notes
Author contributions: G.S., L.R.G., S.E.H., J.d.J., and L.W.G. designed research; G.S., L.E.C., N.M., M.V., and L.W.G. performed research; G.S., L.R.G., N.M., and L.W.G. analyzed data; and G.S., L.R.G., and L.W.G. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PAP, purple acid phosphatase; PP, protein phosphatase.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1XZW).

Footnotes

Patel, K., Lockless, S., Thomas, B. & McKnight, T. (1997) Plant Physiol.III, Suppl., 81 (abstr.).

Footnotes

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