Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

Abstract

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…16,22,24,[27][28][29] Plant PAPs have been extracted from red kidney bean, soybean, sweet potato, duckweed, and tomato. [30][31][32][33][34][35] Most characterized plant PAPs are homodimers with a subunit molecular weight of ∼55 kDa. [30][31][32]35 A heterodimeric intracellular PAP has recently been reported for tomato, 33 with 63 and 57 kDa subunits.…”

The Dual-Targeted Purple Acid Phosphatase Isozyme AtPAP26 Is Essential for Efficient Acclimation of Arabidopsis to Nutritional Phosphate Deprivation

“…Li and coworkers (2002) classified 29 putative PAP genes in Arabidopsis, several of which appear to respond to Pi deficiency. Apart from functioning as nonspecific APases, several PSI plant PAPs (including AtPAP17 and AtPAP26) exhibit alkaline peroxidase activity, indicating their potential alternative role in the metabolism of reactive oxygen species (del Pozo et al, 1999;Bozzo et al, 2002Bozzo et al, , 2004aVeljanovski et al, 2006). Moreover, AtPAP17 was induced by oxidative stress (del Pozo et al, 1999) and ectopic expression of a soybean (Glycine max) mitochondrial PAP (GmPAP3) conferred increased resistance to oxidative stress in transgenic Arabidopsis (Francisca Li et al, 2008).…”

“…A variety of intracellular and secreted APases upregulated by 2Pi plants have been characterized as purple APases (PAPs), which represent a specific APase class characterized by a bimetallic active site that endows them with a pink or purple color in solution (del Pozo et al, 1999;Bozzo et al, 2002Bozzo et al, , 2004aBozzo et al, , 2006Veljanovski et al, 2006;Tran et al, 2010b). Although mammals contain a pair of PAP-encoding genes, vascular plant PAPs belong to a large multigene family.…”

Molecular and Biochemical Characterization of AtPAP15, a Purple Acid Phosphatase with Phytase Activity, in Arabidopsis

“…Not all PAPs exhibit phytase activity; for instance, no phytase activity has been reported for mammalian PAPs. In addition, some plant PAPs, including KbPAP (Cashikar et al, 1997), two secreted tomato PAPs (Bozzo et al, 2002), and an intracellular tomato PAP (Bozzo et al, 2004), lack phytase activity.…”

“…It is intriguing that so many PAP-like genes are required for plant metabolism; this diverse portfolio of PAP-like genes implies differential functions for them. Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphorus compounds (Cashikar et al, 1997;Bozzo et al, 2004;Lung et al, 2008). However, additional biological roles have been reported for some plant PAPs.…”