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J. Biol. Chem., Vol. 281, Issue 39, 29213-29220, September 29, 2006

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Identification of the Cadaverine Recognition Site on the Cadaverine-Lysine Antiporter CadB^*

Waraporn Soksawatmaekhin, Takeshi Uemura, Natsuko Fukiwake, Keiko Kashiwagi, and Kazuei Igarashi¹

From the Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan and the Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan

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 Tyr⁷³, Tyr⁸⁹, Tyr⁹⁰, Glu²⁰⁴, Tyr²³⁵, Asp³⁰³, and Tyr⁴²³ were strongly involved in both uptake and excretion and that Tyr⁵⁵, Glu⁷⁶, Tyr²⁴⁶, Tyr³¹⁰, Cys³⁷⁰, and Glu³⁷⁷ were moderately involved in both activities. Mutations of Trp⁴³, Tyr⁵⁷, Tyr¹⁰⁷, Tyr³⁶⁶, and Tyr³⁶⁸ mainly affected uptake activity, and Trp⁴¹, Tyr¹⁷⁴, Asp¹⁸⁵, and Glu⁴⁰⁸ 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 Arg²⁹⁹ mainly affected excretion, suggesting that Arg²⁹⁹ 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 Cys³⁷⁰ seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys¹²⁵, Cys³⁸⁹, or Cys³⁹⁴ together with Cys³⁷⁰. 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 Cys³⁷⁰. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.

Received for publication, January 25, 2006

^* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; the Sankyo Foundation of Life Science, Japan; and by the Tokyo Biochemical Research Foundation, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-43-226-2871; Fax: 81-43-226-2873; E-mail: iga16077{at}p.chiba-u.ac.jp.

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