Identification of the cadaverine recognition site on the cadaverine-lysine antiporter CadB.
Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) were strongly involved in both uptake and excretion and that Tyr(55), Glu(76), Tyr(246), Tyr(310), Cys(370), and Glu(377) were moderately involved in both activities. Mutations of Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) mainly affected uptake activity, and Trp(41), Tyr(174), Asp(185), and Glu(408) had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the K(m) value. Mutation of Arg(299) mainly affected excretion, suggesting that Arg(299) is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys(370) seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys(125), Cys(389), or Cys(394) together with Cys(370). The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys(370). The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.
Soksawatmaekhin
Waraporn
W
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Uemura
Takeshi
T
Fukiwake
Natsuko
N
Kashiwagi
Keiko
K
Igarashi
Kazuei
K
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000378:metabolism / Q000502:physiology*; D017920:Antiporters / Q000378:metabolism / Q000502:physiology*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002103:Cadaverine / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation
D026905:Amino Acid Transport Systems; D017920:Antiporters; C490242:CadB protein, E Coli; D029968:Escherichia coli Proteins; D001120:Arginine; D008239:Lysine; D003545:Cysteine; D002103:Cadaverine