Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis.
The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) 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 in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.
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Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
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Lanfermeijer
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Konings
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Poolman
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport; D001920:Bradykinin / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005453:Fluorescence; D017353:Gene Deletion; D015151:Immunoblotting; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008074:Lipoproteins; D009842:Oligopeptides; C046506:oligopeptide-binding protein, bacteria; D001920:Bradykinin