Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis

The kinetic properties of wild-type and mutant oligopeptide binding protein s of Lactococcus lactis were determined. To observe the properties of the m utant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide tran sport. Amplified expression of the oppA gene resulted in an 8- to 12-fold i ncrease in OppA protein relative to the wild-type level. The amplified expr ession was paralleled by increased bradykinin binding activity, but had rel atively little effect on the overall transport of bradykinin via Opp. Sever al site-directed mutants were constructed on the basis of a comparison of t he primary sequences of OppA from Salmonella enterica serovar Typhimurium a nd L. lactis, taking into account the known structure of the serovar Typhim urium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-a ffinity peptide bradykinin, Except For OppA(D471R), the mutant OppA protein s displayed highly defective bradykinin uptake, whereas the transport of th e low-affinity substrate KYGK was barely affected. Cells expressing OppA(D4 71R) had a similar K-m for transport, whereas the V-max was increased more than twofold as compared to the wild-type protein. The data are discussed i n the light of a kinetic model and imply that the rate of transport is dete rmined to a large extent by the donation of the peptide from the OppA prote in to the translocator complex.