Functional enhancement by single-residue substitution of Streptomyces coelicolor A3(2) H+-translocating pyrophosphatase.
H(+)-translocating pyrophosphatase converts energy from hydrolysis of pyrophosphate to active H(+) transport across biomembranes. Mutational analysis of Streptomyces coelicolor A3(2) enzyme revealed that amino acid substitution of Phe-388 and Ala-514 altered the enzyme activity. Both residues are located at the interface between the transmembrane domains and cytosolic loops, in which the catalytic domain exists. Systematic amino acid substitution was carried out using the Escherichia coli heterologous expression system. Two of the 38 mutant enzymes, F388Y and A514S, showed a high ratio of H(+)-pump to substrate hydrolysis without decrease in the substrate hydrolysis activity, indicating high energy-coupling efficiency.
Hirono
Megumi
M
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. meg@agr.nagoya-u.ac.jp
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D050505:Mutant Proteins; D043564:Inorganic Pyrophosphatase