Asp(344) and Thr(345) are critical for cation exchange mediated by NhaD, Na(+)/H(+) antiporter of Vibrio cholerae.
The Vc-NhaD is an Na(+)/H(+) antiporter from Vibrio cholerae belonging to a new family of bacterial Na(+)/H(+) antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu(100)Ala, Glu(251)Ala, Glu(342)Ala, and Asp(393)Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp(344)Ala, Asp(344)Asn, and Thr(345)Ala caused a complete loss of both Na(+)/H(+) and Li(+)/H(+) antiport activity whereas the Asp(344)Glu variant exhibited reduced Na(+)/H(+) and Li(+)/H(+) antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na(+)/H(+) antiport. We discuss the possible role of Asp(344) and Thr(345) in the functioning of Vc-NhaD.
Ostroumov
Elena
E
Faculty of Science, Department of Microbiology, University of Manitoba, R3T 2N2 Winnipeg, Manitoba, Canada.
Dzioba
Judith
J
Loewen
Peter C
PC
Dibrov
Pavel
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005798:Genes, Bacterial; D017136:Ion Transport; D008094:Lithium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry; D014170:Transformation, Genetic; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C111423:NhaD protein, Vibrio; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D013912:Threonine; D001224:Aspartic Acid; D008094:Lithium; D012964:Sodium