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.
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Acknowledgments
We thank Taiji Nakae for his valuable discussion and critical reading of the manuscript. We also thank Hatsuhiro Hori for his helpful discussion. This study was supported in part by the Institute for Fermentation, Osaka, a grant from the Japan Science and Technology Agency, and the Sasakawa Scientific Research Grant from The Japan Science Society.
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Communicated by Djamel Drider.
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Kim, S., Ihara, K., Katsube, S. et al. Impact of charged amino acid substitution in the transmembrane domain of l-alanine exporter, AlaE, of Escherichia coli on the l-alanine export. Arch Microbiol 199, 105–114 (2017). https://doi.org/10.1007/s00203-016-1279-4
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DOI: https://doi.org/10.1007/s00203-016-1279-4