title: Role of Asp187 and Gln190 in the Na+/proline symporter (PutP) of Escherichia coli.
abstract: Asp187 and Gln190 were predicted as conserved and closely located at the Na(+) binding site in a topology and homology model structure of Na(+)/proline symporter (PutP) of Escherichia coli. The replacement of Asp187 with Ala or Leu did not affect proline transport activity; whereas, change to Gln abolished the active transport. The binding affinity for Na(+) or proline of these mutants was similar to that of wild-type (WT) PutP. This result indicates Asp187 to be responsible for active transport of proline without affecting the binding. Replacement of Gln190 with Ala, Asn, Asp, Leu and Glu had no effect on transport or binding, suggesting that it may not have a role in the transport. However, in the negative D187Q mutant, a second mutation, of Gln190 to Glu or Leu, restored 46 or 7% of the transport activity of WT, respectively, while mutation to Ala, Asn or Asp had no effect. Thus, side chain at position 190 has a crucial role in suppressing the functional defect of the D187Q mutant. We conclude that Asp187 is responsible for transport activity instead of coupling-ion binding by constituting the translocation pathway of the ion and Gln190 provides a suppressing mutation site to regain PutP functional activity.
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.
authors.forename: Anowarul
authors.initials: A
authors.lastname: Amin
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ando
authors.forename: Shinya
authors.initials: S
authors.lastname: Saijo
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D027981:Symporters / Q000378:metabolism*
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D027981:Symporters; C060609:PutP protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid