Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H+-pyrophosphatase.
The H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly198 showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H+ translocation. Pro189, Asp281, and Val351 in the periplasmic loops were critical for enzyme function. Mutation of Ala357 in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H+-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H+-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.
Hirono
Megumi
M
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Nakanishi
Yoichi
Y
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D000601:Amino Acids, Essential / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm / Q000201:enzymology; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000201:enzymology*
D000601:Amino Acids, Essential; D001426:Bacterial Proteins; D043564:Inorganic Pyrophosphatase