Asp344 and Thr345 are critical for cation exchange mediated by NhaD, Na+/H+ antiporter of Vibrio cholerae

被引：15

作者：

Ostroumov, E ^{[1
]}

Dzioba, J ^{[1
]}

Loewen, PC ^{[1
]}

Dibrov, P ^{[1
]}

机构：

[1] Univ Manitoba, Dept Microbiol, Fac Sci, Winnipeg, MB R3T 2N2, Canada

来源：

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES | 2002年 / 1564卷 / 01期

基金：

加拿大自然科学与工程研究理事会;

关键词：

Na+; H+; antiport; Vibrio cholerae; site-directed mutagenesis; cation-binding site;

D O I：

10.1016/S0005-2736(02)00407-8

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2002 Elsevier Science B.V. All rights reserved.

引用

页码：99 / 106

页数：8

共 27 条

[1] Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].

Altschul, SF ;

Madden, TL ;

Schaffer, AA ;

Zhang, JH ;

Zhang, Z ;

Miller, W ;

Lipman, DJ .

NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402

[2]

CLAROS MG, 1994, COMPUT APPL BIOSCI, V10, P685

[3] Comparative molecular analysis of Na+/H+ exchangers:: a unified model for Na+/H+ antiport? [J].

Dibrov, P ;

Fliegel, L .

FEBS LETTERS, 1998, 424 (1-2) :1-5

[4] Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast [J].

Dibrov, P ;

Young, PG ;

Fliegel, L .

BIOCHEMISTRY, 1998, 37 (23) :8282-8288

[5] MECHANISM OF NA+/H+ EXCHANGE BY ESCHERICHIA-COLI NHAA IN RECONSTITUTED PROTEOLIPOSOMES [J].

DIBROV, PA ;

TAGLICHT, D .

FEBS LETTERS, 1993, 336 (03) :525-529

[6] CALCIUM-TRANSPORT MEDIATED BY NHAA, A NA+/H+ ANTIPORTER FROM ESCHERICHIA-COLI [J].

DIBROV, PA .

FEBS LETTERS, 1993, 336 (03) :530-534

[7]

DZIOBA J, 2000, MOL CELL BIOCHEM, V229, P119

[8] Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na+/H+ antiporter of Escherichia coli [J].

Galili, L ;

Rothman, A ;

Kozachkov, L ;

Rimon, A ;

Padan, E .

BIOCHEMISTRY, 2002, 41 (02) :609-617

[9] Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences [J].

Gerchman, Y ;

Rimon, A ;

Venturi, M ;

Padan, E .

BIOCHEMISTRY, 2001, 40 (11) :3403-3412

[10] CHARACTERIZATION OF A NA+/H+ ANTIPORTER GENE OF ESCHERICHIA-COLI [J].

GOLDBERG, EB ;

ARBEL, T ;

CHEN, J ;

KARPEL, R ;

MACKIE, GA ;

SCHULDINER, S ;

PADAN, E .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (09) :2615-2619

← 1 2 3 →