title: Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay.
abstract: Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP(i). Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni(2+)-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI-TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.
authors.affiliation: Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Moscow119992, Russian Federation. rodina@belozersky.msu.ru
authors.forename: E
authors.initials: E
authors.lastname: Rodina
authors.forename: N
authors.initials: N
authors.lastname: Vorobieva
authors.forename: S
authors.initials: S
authors.lastname: Kurilova
authors.forename: Ju
authors.initials: J
authors.lastname: Mikulovich
authors.forename: J
authors.initials: J
authors.lastname: Vainonen
authors.forename: E-M
authors.initials: EM
authors.lastname: Aro
authors.forename: T
authors.initials: T
authors.lastname: Nazarova
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D043564:Inorganic Pyrophosphatase