Abstract
The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.
Highlights
(bar 2), which inhibited -Ala-Lys-AMCA uptake by 97%, similar inhibition rates of 86, 79, and 79%, respectively, were observed for the three aminocephalosporins cefadroxil, cefalexin, and cephradine at 10 mM concentration
Summary of IC50 values and Ki values Numbers in parentheses indicate the number of independent determinations
Ki values were determined by the equation Ki ϭ IC50/(1 ϩsubstrate/KD) (Cheng and Prusoff [23])
Summary
(bar 2), which inhibited -Ala-Lys-AMCA uptake by 97%, similar inhibition rates of 86, 79, and 79%, respectively, were observed for the three aminocephalosporins cefadroxil (bar 3), cefalexin (bar 4), and cephradine (bar 5) at 10 mM concentration. Cefuroxime (bar 6) and cefamandole (bar 7) showed markedly reduced affinities with modest inhibition of only 21 and 32%, respectively, which results from the lack of an ␣-amino group important for high affinity. Whereas the two aminopenicillins ampicillin (bar 8) and amoxicillin (bar 9, 5 mM concentration) seemed not to serve as substrates, the angiotensin I-converting enzyme inhibitors captopril (bar 10) and enalapril (bar 11) showed only low affinity type inhibition o
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