title: Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.
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.
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.
authors: Seryoung
authors: S
authors: Kim
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.
authors: Kohei
authors: K
authors: Ihara
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.
authors: Satoshi
authors: S
authors: Katsube
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.
authors: Tasuke
authors: T
authors: Ando
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan.
authors: Emiko
authors: E
authors: Isogai
authors: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, 981-8555, Japan. hiroshi.yoneyama.a4@tohoku.ac.jp.
authors: Hiroshi
authors: H
authors: Yoneyama
mesh_terms: D000409:Alanine / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001692:Biological Transport; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains
chemical_list: C560941:AlaE protein, E coli; D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D001224:Aspartic Acid; D001120:Arginine; D003545:Cysteine; D000409:Alanine
keywords: AlaE; Amino acid exporter; Escherichia coli; L-Alanine