title: The pivotal twin histidines and aromatic triad of the Escherichia coli ammonium channel AmtB can be replaced.
abstract: In Escherichia coli, each subunit of the trimeric channel protein AmtB carries a hydrophobic pore for transport of NH(4)(+) across the cytoplasmic membrane. Positioned along this substrate conduction pathway are two conserved elements--a pair of hydrogen-bonded histidines (H168/H318) located within the pore itself and a set of aromatic residues (F107/W148/F215) at its periplasmic entrance--thought to be critical to AmtB function. Using site-directed mutagenesis and suppressor genetics, we examined the requirement for these elements in NH(4)(+) transport. This analysis shows that AmtB can accommodate, by either direct substitution or suppressor generation, acidic residues at one or both positions of the H168/H318 twin-histidine site while retaining near wild-type activity. Similarly, study of the F107/W148/F215 triad indicates that good-to-excellent AmtB function is preserved upon individual and simultaneous replacement of these aromatic amino acids with aliphatic residues. Our findings lead us to conclude that these elements and their component parts are not required for AmtB function, but instead serve to optimize its performance.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA. jahall@ucsd.edu
authors.forename: Jason A
authors.initials: JA
authors.lastname: Hall
authors.forename: Sydney
authors.initials: S
authors.lastname: Kustu
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000596:Amino Acids; C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D000644:Quaternary Ammonium Compounds; D006639:Histidine