This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Degen, O. Articles by Eitinger, T. Search for Related Content PubMed PubMed Citation Articles by Degen, O. Articles by Eitinger, T.

 Previous Article  |  Next Article 

Journal of Bacteriology, July 2002, p. 3569-3577, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3569-3577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II

Olaf Degen and Thomas Eitinger^*

Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, 10115 Berlin, Germany

Received 20 January 2002/ Accepted 3 April 2002

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. Two histidine residues in a Ni²⁺ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co²⁺ and Ni²⁺ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni²⁺ uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni²⁺ and Co²⁺ with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni²⁺ uptake capacity and conferred to the variant the ability to transport Co²⁺. 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.

---

* Corresponding author. Mailing address: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestraße 117, 10115 Berlin, Germany. Phone: 49-30-2093-8103. Fax: 49-30-2093-8102. E-mail: thomas.eitinger{at}rz.hu-berlin.de.

---

Journal of Bacteriology, July 2002, p. 3569-3577, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3569-3577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Rodionov, D. A., Hebbeln, P., Gelfand, M. S., Eitinger, T. (2006). Comparative and Functional Genomic Analysis of Prokaryotic Nickel and Cobalt Uptake Transporters: Evidence for a Novel Group of ATP-Binding Cassette Transporters. J. Bacteriol. 188: 317-327 [Abstract] [Full Text]  
• Eitinger, T. (2004). In Vivo Production of Active Nickel Superoxide Dismutase from Prochlorococcus marinus MIT9313 Is Dependent on Its Cognate Peptidase. J. Bacteriol. 186: 7821-7825 [Abstract] [Full Text]