New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

•A label-free transport assay has been developed to determine substrate specificities of proton-coupled peptide transporters.•Alanine was found to be a substrate for YjdL, but not for YdgR and YhiP.•Di- and trialanine were substrates for all three transporters.•Tetraalanine was found to be a substrate for YdgR and YhiP but not for YjdL.•The preference for a dipeptide substrate with a C-terminal lysine was confirmed through saturation kinetics for YjdL.

Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.