Impact of charged amino acid substitution in the transmembrane domain of l-alanine exporter, AlaE, of Escherichia coli on the l-alanine export

Seryoung Kim, Kohei Ihara, Satoshi Katsube, Tasuke Ando, Emiko Isogai, Hiroshi Yoneyama

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The Escherichia coli alaE gene encodes the l-alanine exporter, AlaE, that catalyzes active export of l-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient l-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to l-alanyl-l-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of l-alanine. To elucidate the mechanism by which AlaE exports l-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the l-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in l-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on l-alanine export.

Original languageEnglish
Pages (from-to)105-114
Number of pages10
JournalArchives of Microbiology
Volume199
Issue number1
DOIs
Publication statusPublished - 2017 Jan 1

Keywords

  • AlaE
  • Amino acid exporter
  • Escherichia coli
  • l-Alanine

ASJC Scopus subject areas

  • Microbiology
  • Biochemistry
  • Molecular Biology
  • Genetics

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