J. Biochem, 1997, Vol. 121, No. 4 661-670
© 1997 Japanese Biochemical Society
research-article |
Identification and Characterization of Functional Residues in a Na+/H+ Antiporter (NhaA) from Escherichia coli by Random Mutagenesis1
*Department of Biotechnology, Faculty of Engineering Sciences, Okayama University Okayama 700
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University Okayama 700
2To whom correspondence should be addressed at the present address: Research and Development Sub-Division, Pharmaceutical Division, Ciba-Geigy Japan Ltd., 10-66 Miyuki-cho, Takarazuka 665. Phone: +81-797-74-2367, Fax: +81-797-74-2530, E-mail: tnoumi{at}po.iijnet.or.jp
Forty-one mutants were isolated by means of random PCR mutagenesis of the Escherichia coli Na+/H+ antiporter (nhaA), which could not support the growth of a nhaAnhaB mutant (HIT 
AB ) on plates containing 0.15 M LiCl (pH 7.5) or 0.65 M NaCl (pH 8.0). Most of the mutants were sensitive to both NaCl and LiCl, or to LiCl alone. DNA sequencing revealed that twelve of the mutants had single amino acid substitutions. All the mutations, except for H225P, were of the conserved residues of NhaA homologues and located in the putative transmembrane helices. The Na+/H+ and Li+/H+ antiporter activities of the mutant NhaA were measured with everted membrane vesicles: eight of the mutants lost both antiporter activities completely under all pH conditions examined. Although both D133A and L138P retained low Li+/H + antiporter activity, D133A lost Na+/H+ antiporter activity, while L138P retained normal Na+/H+ antiporter activity at pH 7.0 and 8.0. Interestingly, at pH 8.5, L138P no longer showed any Li+/ H+ antiporter activity. H225P retained relatively high antiporter activities, although their pH dependence was altered. These observations supported the previous indication that His-225 is part of the pH sensor [Gerchman, Y. et al. (1993) Proc. Natl. Acad. Set USA 90, 1212-1216]. L73R exhibited about 20% each of the activities only at pH 8.0, and showed a similar pH dependence to H225P in both Na+/H+ and Li+/H+ antiport. Therefore, in addition to His-225, Leu-73, and/or its vicinity may also contribute to the pH sensing.
1This study was supported partly by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to T.N. and H.K.
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