Endopolyphosphatases for Long Chain Inorganic Polyphosphate in Yeast and Mammals

Abstract

Whereas exopolyphosphatases have been purified from yeast and a variety of bacteria, this is the first report characterizing endopolyphosphatases that act on long chain inorganic polyphosphate (polyP). The activity from Saccharomyces cerevisiae, localized in vacuoles, has been purified to homogeneity from a strain that possesses vacuolar proteases. The endopolyphosphatase is a dimer of 35-kDa subunits. Distributive action on polyP 750 produces shorter chains to a limit of about polyP 60 , as well as the more abundant release of polyP 3 ; the K m for polyP 750 is 185 nM. Endopolyphosphatases have been identified in a wide variety of sources, except for most eubacteria tested. The activity has been partially purified from rat and bovine brain where its abundance is about 10 times higher than in other tissues but less than 1 ⁄10 that of yeast; the limit product of digestion of the partially purified brain enzyme is polyP 3 .Inorganic polyphosphates (polyP) 1 are linear polymers of orthophosphate residues linked by high energy phosphoanhydride bonds. Likely prevalent in prebiotic evolution (1) polyP has been found in all organisms ranging from bacteria to mammals (2). The ubiquitous occurrence of polyP suggests multiple roles depending on the species, cell, subcellular localization, and physiological state.Our approach toward understanding the functions of polyP has been to identify and isolate the enzymes that synthesize and degrade polyP. Such enzymes have been identified in a variety of microorganisms. PolyP kinase, the enzyme that catalyzes the reversible transfer of the terminal phosphate from ATP to synthesize polyP, has been purified to homogeneity in several bacteria (3)(4)(5). Exopolyphosphatases that catalyze the hydrolysis of terminal phosphates from polyP have been purified from Escherichia coli (6), Corynebacterium xerosis (7), and Saccharomyces cerevisiae (8,9). Phosphotransferases that transfer a phosphate from polyP to AMP (10), NAD (11), glucose (12), and 1,3-diphosphoglycerate (13) have also been described. Endopolyphosphatases (PPN) also called polyP depolymerases or polyphosphorylases catalyze the non-processive cleavage of polyP to release intermediate-size chains during the course of the reaction. PPN are the least studied of the polyP-metabolizing enzymes and have been reported in species of Penicillium and Aspergillus and in S. cerevisiae (14 -16).During the course of investigating polyP metabolism in a variety of cells, we discovered PPN activities in organisms from archae to mammals but little or none among eubacteria. In this report, we describe the purification and characteristics of PPN from S. cerevisiae, where it is most abundant, using a mutant strain in which a major exopolyPase activity has been deleted (17). EXPERIMENTAL PROCEDURESCells and Tissues-S. cerevisiae CRX (17) was grown in YPD medium (1% yeast extract, 2% tryptone, 2% glucose) at 30°C to an A 600 of 14.5. The harvested cells were resuspended in an equal volume of 50 mM Tris-HCl, pH 7.5, 10% sucrose, frozen in liquid ni...

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“…The enzyme that is primarily responsible for reversible poly-P synthesis in Escherichia coli is poly-P kinase 1 (PPK1), which catalyzes the polymerization of the ␥ phosphate of ATP into a poly-P chain (5). Poly-P can also be hydrolyzed to P i either by exopolyphosphatase (PPX) (6) or by endopolyphosphatase (PPN) (7,8). In E. coli, the genes for PPK1 and PPX are under the control of a common promoter.…”

“…Poly-P is ubiquitous, having been found in all organisms (archaea, bacteria, fungi, plants, insects, and mammals) (1)(2)(3). Several poly-P-metabolizing enzymes have been purified and characterized, and the corresponding genes have been cloned and deleted (3)(4)(5)(6)(7)(8). The enzyme that is primarily responsible for reversible poly-P synthesis in Escherichia coli is poly-P kinase 1 (PPK1), which catalyzes the polymerization of the ␥ phosphate of ATP into a poly-P chain (5).…”

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks

“…Exopolyphosphatases have been found in prokaryotes and eukaryotes and, although in bacteria these enzymes mostly hydrolyze high molecular weight polyphosphates (Kumble & Kornberg, 1996), at least some of the enzymes from Saccharomyces cerevisiae and Leishmania major are more active in hydrolyzing short chain polyphosphates, such as polyphosphate 3 (Kumble & Kornberg, 1996;Rodrigues et al, 2002). Exopolyphosphatase from Escherichia coli requires divalent cations and K + for maximum activity, while exopolyphosphatase from yeast only requires divalent cations (Lichko et al, 2003).…”