Enzymes of Polyphosphate Metabolism in Yeast: Properties, Functions, Practical Significance.

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.

The acid phosphatase Pho5 of Saccharomyces cerevisiae is not involved in polyphosphate breakdown.

Inorganic polyphosphate is involved in architecture and functioning of yeast cell wall. The strain of Saccharomyces cerevisiae constitutively overexpressing acid phosphatase Pho5 was constructed for studying the Pho5 properties and its possible participation in polyphosphate metabolism. The parent strain was transformed by the vector carrying the PHO5 gene under a strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae. The culture liquid and biomass of transformant strain contained approximately equal total acid phosphatase activity. The levels of acid phosphatase activity associated with the cell wall and culture liquid increased in the transformant strain compared to the parent strain ~ 10- and 20-fold, respectively. The Pho5 preparation (specific activity of 46 U/mg protein and yield of 95 U/L) was obtained from culture liquid of overproducing strain. The overproducing strain had no changes in polyphosphate level. The activity of Pho5 with long-chained polyP was negligible. We concluded that Pho5 is not involved in polyphosphate metabolism. Purified Pho5 showed a similar activity with p-nitrophenylphosphate, ATP, ADP, glycerophosphate, and glucose-6-phosphate. The substrate specificity of Pho5 and its extracellular localization suggest its function: the hydrolysis of organic compounds with phosphoester bonds at phosphate limitation.

Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae.

Inorganic polyphosphate performs many regulatory functions in living cells. The yeast exopolyphosphatase PPN1 is an enzyme with multiple cellular localization and probably variable functions. The Saccharomyces cerevisiae strain with overexpressed PPN1 was constructed for large-scale production of the enzyme and for studying the effect of overproduction on polyphosphate metabolism. The ΔPPN1 strain was transformed by the vector containing this gene under a strong constitutive promoter of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae. Exopolyphosphatase activity in the transformant increased 28- and 11-fold compared to the ΔPPN1 and parent strains, respectively. The content of acid-soluble polyphosphate decreased ∼6-fold and the content of acid-insoluble polyphosphate decreased ∼2.5-fold in the cells of the transformant compared to the ΔPPN1 strain. The recombinant enzyme was purified. The substrate specificity, cation requirement, and inhibition by heparin were found to be similar to native PPN1. The molecular mass of a subunit (∼33 kD) and the amino acid sequence of the recombinant enzyme were the same as in mature PPN1. The recombinant enzyme was localized mainly in the cytoplasm (40%) and vacuoles (20%). The overproducer strain had no growths defects under phosphate deficiency or phosphate excess. In contrast to the parent strains accumulating polyphosphate, the transformant accumulated orthophosphate under phosphate surplus.