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

Abstract

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

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

“…Nickel incorporation in Escherichia coli hydrogenases also requires a GTP-binding accessory protein, HypB (11). The protein sequences of a number of microbial nickel uptake systems have been described (12)(13)(14), but to date no structural information has been obtained. Nickel uptake by bacteria is of interest for bioremediation and water purification as well as its biological function and involvement in pathogenesis.…”

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

“…Overall, these data suggest that these four transmembrane domains are directly involved in binding and translocation of Ni ions, likely constituting a pore through which Ni could pass. The first motif, HX 5 DH, is highly similar to that described in the NiCoT permease family as essential for Ni uptake, 11 whereas the second motif, FHGX[AV]HGXE, is very different and may define a novel way for Ni ions to cross the membrane. It is not known how these motifs participate in the forming of a pore, and we have no information about the number of HupE monomers that could be involved in such a pore.…”

“…9,10 These two proteins share a topology with eight transmembrane domains, 11,12 and two conserved histidine-rich motifs critical for metal uptake (GX 2 HX 4 DH and GX 2 FX 2 GH). 11,13 The UreH group includes Ni-specific permeases whose corresponding genes are associated with gene clusters for urease or Ni-superoxide dismutase in the genome of Bacillus and also in several proteobacteria and cyanobacteria. 5,14 Finally, members of the HupE/UreJ family are widespread among bacteria, and are usually encoded within hydrogenase or urease gene clusters.…”

Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

“…In this direction, the involvement of conserved transmembrane His, Glu, and Ser in Fe 2ϩ binding by Arabidopsis IRT1 (a member of the ZIP family of transporters) has been proposed (28). A similar role has been indicated for transmembrane His and carboxylic amino acids in the case of NixA-type Ni 2ϩ permeases (29,30) and the DMT1 Fe 2ϩ transporters (31). In contrast, the transmembrane cation binding sites have been well described for the Ca-ATPase and Na,K-ATPase, extensively characterized P IItype cation pumps (32)(33)(34)(35)(36)(37)(38).…”