Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic sys tem to degrade exogenous proteins such as caseins. The extracellular cell w all-attached proteinase PrtP and the oligopeptide transport system Opp medi ate the first two steps in the utilization of caseins, beta-Casein is degra ded by PrtP to fragments of 5-30 amino acid residues, and only a limited nu mber of peptides are selected from this pool for uptake via Opp. To study t he specificity of Opp and the kinetics of peptide uptake in L. lactis in de tail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (i ii) iodinated KYGK was used as the reporter peptide; (iv) libraries of pept ides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was d etermined, and the uptake of these peptides was followed by high-performanc e liquid chromatography (HPLC). These studies indicate that (i) the Opp sys tem can transport a broad range of peptides from 4 up to at least 18 residu es with very little preference for particular side chains and (ii) the kine tics of peptide uptake differ for different substrates tested. Whereas clas s I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the lev el of uptake of the majority of peptides (class II) increases sigmoidally w ith concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.