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Journal of Biochemistry Advance Access originally published online on July 23, 2009
Journal of Biochemistry 2009 146(5):617-621; doi:10.1093/jb/mvp114
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Rapid Communications

Functional Enhancement by Single-residue Substitution of Streptomyces coelicolor A3(2) H+-translocating Pyrophosphatase

Megumi Hirono* and Masayoshi Maeshima

Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-52-789-4096, E-mail: meg{at}agr.nagoya-u.ac.jp

Received June 30, 2009; Accepted July 3, 2009


  Abstract

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.

Key Words: energy coupling, H+-pyrophosphatase, proton pump, site-directed mutagenesis, structure–function relationship

Abbreviations: H+-PPase, H+-pyrophosphatase; ScPP, Streptomyces coelicolor A3(2) H+-PPase; PPi, pyrophosphate


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