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 system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, 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; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, 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 determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.
Detmers
F J
FJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Kunji
E R
ER
Lanfermeijer
F C
FC
Poolman
B
B
Konings
W N
WN
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002364:Caseins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides; C046506:oligopeptide-binding protein, bacteria; C037230:oligopeptide permease, Bacteria; D010450:Endopeptidases