HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. . NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.In the functional phylogenetic transporter classification system developed by Saier (26,27), the family with the code TC 2.A.52 is described as the nickel/cobalt transporter (NiCoT) family. The family comprises structurally related membrane proteins in gram-negative and gram-positive bacteria, in archaea, and in fungi. An eight-helix structure, conserved signatures containing charged residues in transmembrane domains (TMDs), and a large hydrophilic loop connecting TMDs IV and V are common features of the members of this family (reviewed in references 5, 8, and 9). High-affinity Ni 2ϩ uptake to provide the metal ion for incorporation into Ni-containing metalloenzymes (see reference 14 for a review) is the physiological role of the NiCoTs of Ralstonia eutropha (HoxN), Helicobacter pylori (NixA), and the fission yeast Schizosaccharomyces pombe. Despite the family name, HoxN acts as a selective Ni 2ϩ permease and does not transport Co 2ϩ (4). NixA activity in the human pathogen H. pylori is essential to virulence, since nickel-containing urease is a central pathogenicity determinant in this species. Site-directed mutagenesis has been employed to localize residues and motifs in HoxN (10) and NixA (12) that cannot be altered without a dramatic or complete loss of activity. These studies uncovered the relevance of a strongly conserved segment (GLR/KHAV/ FDADHI/LAAI) in TMD II that can be considered a signature sequence for NiCoTs. NhlF, the NiCoT of the gram-positive Rhodococcus rhodochrous J1, provides Co 2ϩ ion for incorporation into Co 2ϩ -containing nitrile hydratases (17), industrial catalysts that contain a non-corrin cobalt metallocenter (reviewed in reference 16). In contrast to HoxN, NhlF is literally a NiCoT and transports Ni 2ϩ and Co 2ϩ with high affinity (4). Experimental analysis of NiCoT activity is difficult. These permeases transport their substrates with very high affinity but extremely low capacity. For reproducible measurement of HoxN activity, hoxN has been expressed in Escherichia coli and metal uptake has been analyzed during growth in complex medium (33). At a 63 Ni 2ϩ concentration of 500 nM in the growth medium, the resulting cellular 63 Ni content was in the range of 25 to 50 pmol per mg of protein, corresponding to approximately 2,500 to 5,000 Ni 2ϩ ions per cell. T...
nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. 57CO2+ and 63Ni2+ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport 57CO2+, but mediated 63Ni2+ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni2+. In contrast, NhlF transported both 57CO2+ and 63Ni2+ ion. NhlF-mediated 63Ni2+ uptake was markedly reduced in the presence of CO2+, while 57CO2+ uptake was only slightly lower in the presence of Ni2+. These results indicate different affinities of NhlF for CO2+ and Ni2+ and identified CO2+ ion as the preferred substrate.
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 wildtype 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.Alcaligenes eutrophus H16, a member of the  division of the proteobacteria, can utilize various organic compounds or molecular hydrogen as energy sources. Hydrogen oxidation is catalyzed by two metalloenzymes: a cytoplasmic NAD ϩ -reducing hydrogenase and a membrane-bound, electron transport-coupled hydrogenase. Both enzymes belong to the family of [NiFe] hydrogenases (reviewed in Ref. 1). A nickel-dependent urease allows the organism to grow on urea as a nitrogen source.Uptake of nickel ion in sufficient amounts is a prerequisite for the synthesis of nickel-containing enzymes. Microbial nickel uptake is mediated by nonspecific transport systems for divalent cations and by specific systems with a high affinity for Ni 2ϩ . In natural environments the concentration of Ni 2ϩ is generally very low compared with other divalent cations like Mg 2ϩ . Since nonspecific nickel transport is competitively inhibited by a number of divalent cations, this mode of uptake is not suited for meeting the cellular nickel requirements (reviewed in Refs. 2 and 3). Molecular analyses of Ni 2ϩ -specific transport systems of a few bacteria containing [NiFe] hydrogenases and/or ureases showed that two different types of membrane transporters are responsible for high affinity uptake of the transition metal (reviewed in Ref. 3). The best studied Ni 2ϩ
The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases. These uptake systems transport Ni 2؉ ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations. An S. pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in 63
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