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The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold.

ABSTRACT: Phosphatases function in the production, transport and recycling of inorganic phosphorus, which is crucial for cellular metabolism and bioenergetics, as well as in bacterial killing, since they are able to generate reactive oxygen species via Fenton chemistry. Diphosphonucleotide phosphatase/phosphodiesterase (PPD1), a glycoprotein plant purple acid phosphatase (PAP) from yellow lupin seeds, contains a bimetallic Fe-Mn catalytic site which is most active at acidic pH. Unlike other plant PAPs, PPD1 cleaves the pyrophosphate bond in diphosphonucleotides and the phosphodiester bond in various phosphodiesters. The homohexameric organization of PPD1, as revealed by a 1.65?Å resolution crystal structure and confirmed by solution X-ray scattering, is unique among plant PAPs, for which only homodimers have previously been reported. A phosphate anion is bound in a bidentate fashion at the active site, bridging the Fe and Mn atoms in a binding mode similar to that previously reported for sweet potato PAP, which suggests that common features occur in their catalytic mechanisms. The N-terminal domain of PPD1 has an unexpected and unique fibronectin type III-like fold that is absent in other plant PAPs. Here, the in vitro DNA-cleavage activity of PPD1 is demonstrated and it is proposed that the fibronectin III-like domain, which 'overhangs' the active site, is involved in DNA selectivity, binding and activation. The degradation of DNA by PPD1 implies a role for PPD1 in plant growth and repair and in pathogen defence.

SUBMITTER: Antonyuk SV

PROVIDER: S-EPMC4062093 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold.

Antonyuk Svetlana Vladimirovna SV Olczak Mariusz M Olczak Teresa T Ciuraszkiewicz Justyna J Strange Richard William RW

IUCrJ 20140228 Pt 2

Phosphatases function in the production, transport and recycling of inorganic phosphorus, which is crucial for cellular metabolism and bioenergetics, as well as in bacterial killing, since they are able to generate reactive oxygen species via Fenton chemistry. Diphosphonucleotide phosphatase/phosphodiesterase (PPD1), a glycoprotein plant purple acid phosphatase (PAP) from yellow lupin seeds, contains a bimetallic Fe-Mn catalytic site which is most active at acidic pH. Unlike other plant PAPs, PP ...[more]

PMID: 25075326

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Project description:The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.

Dataset Information

The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold.

Publications

The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold.

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