Importance of conserved acidic residues in mntH, the Nramp homolog of Escherichia coli.
A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of (54)Mn(2+) uptake with a V(max) value of approximately 10% of wild-type and a slightly elevated K(m) value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn(2+). Other residues such as Glu-102 and Asp238 may play a role in the release of Mn(2+) to the cytoplasm or may be involved in maintaining secondary structure.
Haemig
H A H
HA
Department of Genetics, Cell Biology and Development, the Biotechnology Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Brooker
R J
RJ
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics*; D001693:Biological Transport, Active / Q000235:genetics / Q000502:physiology; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C492412:MntH protein, E coli; D008345:Manganese