title: Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino 142290 Russia.
authors.forename: Nadeshda
authors.initials: N
authors.lastname: Andreeva
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino 142290 Russia.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Trilisenko
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino 142290 Russia. Electronic address: alla@ibpm.pushchino.ru.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kulakovskaya
authors.affiliation: Bioengineering Centre, Russian Academy of Sciences, pr. Shestidesyatiletiya Oktyabrya 7-1, Moscow 117312, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Dumina
authors.affiliation: Bioengineering Centre, Russian Academy of Sciences, pr. Shestidesyatiletiya Oktyabrya 7-1, Moscow 117312, Russia.
authors.forename: Michail
authors.initials: M
authors.lastname: Eldarov
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000201:enzymology; D015870:Gene Expression; D013058:Mass Spectrometry; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology
chemical_list: D010710:Phosphates; D011122:Polyphosphates; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D017766:Acid Anhydride Hydrolases; C000598747:Ppn1 protein, S cerevisiae; C020137:exopolyphosphatase
keywords: Constitutive promoter; Exopolyphosphatase; PPN1 gene; Phosphate surplus; Polyphosphate; Saccharomyces cerevisiae