title: Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus.
abstract: NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.
authors.affiliation: Department of Genomics and Proteomics, Advanced Science Research Center, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Otsuka
authors.forename: Makoto
authors.initials: M
authors.lastname: Yasuda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Morita
authors.forename: Chie
authors.initials: C
authors.lastname: Otsuka
authors.forename: Tomofusa
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Omote
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Moriyama
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001693:Biological Transport, Active / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009643:Norfloxacin / Q000378:metabolism; D017354:Point Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013997:Time Factors; D014736:Vibrio parahaemolyticus / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; C114619:NorM protein, bacteria; D012964:Sodium; D004996:Ethidium; D009643:Norfloxacin
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