Investigation of the metal binding site of MntH, a divalent metal cation/hydrogen(+) transporter of Escherichia coli.

摘要

Site-directed mutagenesis of E.coli MntH was performed on residues that are highly conserved among the Nramp (Natural Resistance Associated Macrophage Protein) family to which MntH belongs. Negatively charged residues (Asp-34, Glu-102, Asp-109, and Glu-112, Glu-154, and Asp-238) conserved within the family were of particular interest for their possible roles in transport of divalent metal cations across the inner membrane. In addition, transmembrane domain six contains a number of highly conserved residues, which were studied with respect to metal transport. Each mutation to MntH was characterized by transport ability, protein expression, and kinetic properties. The results led to a model for the metal binding site within MntH. The proposed site includes the negatively charged residues Asp-34, Asp-109, Glu-112 and the highly conserved residues Pro-35, Gly-36, Asn-37, and Gly-115. This is the first model proposed for the metal binding site for any member of the Nramp family.; As a secondary approach to studying the metal binding site, a method was developed to purify the E.coli MntH protein for use in biophysical studies. A third approach involved constructing chimeric transporters between E.coli MntH and Lactobacillus plantarum MntH---copy1 which has a different metal specificity.