Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase |
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Authors: | Megumi Hirono |
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Affiliation: | Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan |
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Abstract: | 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. |
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Keywords: | DTT, dithiothreitol EGTA, ethylene glycol bis(2-aminoethylether)-N,N,N&prime ,N&prime -tetra-acetic acid H+-PPase, H+-translocating inorganic pyrophosphatase IPTG, isopropyl-1-thio-β-galactopyranoside LB, Luria Broth PPi, pyrophosphate ScPP, Streptomyces coelicolor A3(2) H+-PPase sScPP, synthetic ScPP gene TM, transmembrane domain WT, wild type |
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