Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus.
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.
Otsuka
Masato
M
Department of Genomics and Proteomics, Advanced Science Research Center, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Yasuda
Makoto
M
Morita
Yuji
Y
Otsuka
Chie
C
Tsuchiya
Tomofusa
T
Omote
Hiroshi
H
Moriyama
Yoshinori
Y
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*
D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; C114619:NorM protein, bacteria; D012964:Sodium; D004996:Ethidium; D009643:Norfloxacin
Clin Infect Dis. 1998 Aug;27 Suppl 1:S32-41
9710669
Antimicrob Agents Chemother. 1998 Jul;42(7):1778-82
9661020
J Bacteriol. 2000 Dec;182(23):6694-7
11073914
Microbiol Mol Biol Rev. 2000 Dec;64(4):672-93
11104814
Antimicrob Agents Chemother. 2001 Dec;45(12):3341-6
11709306
J Bacteriol. 2002 Jan;184(2):572-6
11751837
Antimicrob Agents Chemother. 2002 Sep;46(9):3050-3
12183269
Eur J Biochem. 2003 Mar;270(5):799-813
12603313
Diabetes. 2003 Aug;52(8):2066-74
12882924
Science. 2003 Aug 1;301(5633):610-5
12893935
Science. 2003 Aug 1;301(5633):616-20
12893936
Microbiol Immunol. 2003;47(6):419-27
12906102
J Bacteriol. 2004 Jan;186(1):262-5
14679249
Microbiol Immunol. 2003;47(12):937-43
14695443
Appl Environ Microbiol. 2004 Feb;70(2):693-703
14766544
J Biochem. 1979 Jul;86(1):225-30
39066
Gene. 1991 Jun 15;102(1):67-70
1864511
J Biol Chem. 1991 Nov 25;266(33):22141-6
1834667
J Biol Chem. 1992 Apr 15;267(11):7490-8
1313805
Mol Microbiol. 1999 Jan;31(1):394-5
9987140