TY  - BOOK
AU  - Hirono,M.
AU  - Nakanishi,Y.
AU  - Maeshima,M.
TI  - Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2)H+-pyrophosphatase
KW  - ENERGY COUPLING
KW  - H+-PYROPHOSPHATASE
KW  - RANDOM MUTAGENESIS
KW  - PROTON PUMP
N2  - 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
UR  - https://drive.google.com/file/d/13j3eiG0UQc7UJv3y-Tot27JGCUKXU2wJ/view?usp=drivesdk
ER  -