Kinetic and Mutational Analyses of the Major Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae

Abstract

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase -bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase. Linear inorganic polyphosphates (polyP)3 comprising chains of tens to hundreds of phosphate units are conserved in all cells, and thus are possible agents of evolution from prebiotic times (1, 2). In eukaryotes, they account for up to 20% of dry cell weight (1, 2). Recent evidence indicates that rather than simply being a store of phosphate and energy, polyP are required for bacterial responses to a variety of stress and stringency conditions, as well as for virulence of some pathogens (3-5). PolyP are additionally involved in blood clotting (6) and proliferation of mammary cancer cells (7).PolyP are synthesized by polyphosphate kinase, and hydrolyzed by exo-and endopolyphosphatases. Exopolyphosphatase (PPX) processively releases the terminal phosphate groups from polyP formed by Ն3 phosphate residues. Based on the primary structure, PPXs are classified into two types, whose prototypes are PPXs from yeast (Saccharomyces cerevisiae) cytosol and Escherichia coli. Yeast-type PPX, reported in fungi and protozoa, belongs to the superfamily of DHH phosphoesterases (named after the conserved Asp-His-His motif) (8), whereas E. coli-type PPX is present in Eubacteria and Archaea and belongs to a sugar kinase/actin/hsp-70 superfamily (9). Genes of both yeast-type PPXs (from S. cerevisiae and Leishmania major) and E. coli type PPXs (from E. coli, Aquifex aeolicus and Sulfolobus solfataricus) have been expressed in E. coli (10 -14), and the structures of S. cerevisiae (15), E. coli (16), 4 and A. aeolicus (E. coli type) (18) PPXs have been determined. The overall folds of the two PPX families are dissimilar. Specifically, the subunits comprise two domains in yeast-type PPX and four domains in E. coli-type PPX. However, in both families, the active site is located between the domains connected by a flexible linker. Interestingly, yeast and A. aeolicus PPXs are monomeric proteins (15, 18 -20), whereas E. coli PPX is dimeric (10, 16). 4 Interestingly, the yeast cell contains up to five different exopolyphosphatases in different compartments (22).The overall structure of yeast cytosolic PPX (scPPX) bears a striking similarity to that of the well characterized family II pyrophosphatase (PPase) (23, 24), a DHH phosphoesterase that catalyzes a similar reaction with pyrophosphate, the shortest polyphosphate. Despite only 12-17% sequence identity, 11 of the total 14 polar residues in the active site of family II PPase are conserved in all yeast-type PPXs, and two more are conserved in most of the enzymes (Fig. 1A), implying an evolutionary relationship. M...

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Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

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Human Metastasis Regulator Protein H-Prune is a Short-Chain Exopolyphosphatase

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