title: Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties.
abstract: H(+)-translocating pyrophosphatases (H(+)-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H(+)-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H(+)-PPase in Escherichia coli. The H(+)-PPase from E. coli membranes was active at a relatively high pH, stable up to 50 degrees C, and sensitive to N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K(+), which was less than the stimulation observed with plant vacuolar H(+)-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K(+) requirement of H(+)-PPases, did not alter its K(+) dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H(+)-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures.
authors.affiliation: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University.
authors.forename: Megumi
authors.initials: M
authors.lastname: Hirono
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Mimura
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maeshima
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D043564:Inorganic Pyrophosphatase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D043564:Inorganic Pyrophosphatase; D011188:Potassium