TAILIEUCHUNG - Báo cáo khoa học: DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

The genome ofEscherichia colicontains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been character-ized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgHandyjdLgenes have remained unknown. | ễFEBS Journal DtpB YhiP and DtpA TppB YdgR are prototypical proton-dependent peptide transporters of Escherichia coli Daniel Harder1 Jurgen Stolz1 Fabio Casagrande2 Petr Obrdlik3 Dietmar Weitz1 Dimitrios Fotiadis2 and Hannelore Daniel1 1 Molecular Nutrition Unit TechnicalUniversity of Munich Freising Germany 2 . Muller Institute for StructuralBiology Biozentrum University of Basel Switzerland 3 IonGate Biosciences GmbH Frankfurt Germany Keywords E. coli peptide PTR TppB transport Correspondence H. Daniel Molecular Nutrition Unit TechnicalUniversity of Munich Am Forum 5 D-85350 Freising Germany Fax 49 8161713999 Tel 49 8161713400 E-mail daniel@ Present address Institute of Biochemistry and Molecular Medicine University of Berne Switzerland Received 18 February 2008 revised 20 March 2008 accepted 23 April 2008 doi The genome of Escherichia coli contains four genes assigned to the peptide transporter PTR family. Of these only tppB ydgR has been characterized and named tripeptide permease whereas protein functions encoded by the yhiP ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine Gly-Sar with an apparent affinity constant of mM. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion suggesting H -dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together these

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