Expression of Functional Streptomyces coelicolor H+-Pyrophosphatase and Characterization of Its Molecular Properties.

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°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. [ABSTRACT FROM PUBLISHER]