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Volume 272, Number 27, Issue of July 4, 1997 pp. 17139-17144
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

A Ni²⁺ Binding Motif Is the Basis of High Affinity Transport of the Alcaligenes eutrophus Nickel Permease

(Received for publication, October 29, 1996, and in revised form, April 1, 1997)

Thomas Eitinger , Lutz Wolfram , Olaf Degen ^¶ and Carolin Anthon ^¶

From the  Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestraße 117, D-10115 Berlin and ^¶ Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, D-14195 Berlin, Germany

Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated. Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T. (1995) J. Bacteriol. 177, 1840-1843). Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake. This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges. Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E. coli system. The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease. These mutations were reintroduced into the A. eutrophus wild-type strain. Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion.

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