Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis

Abstract

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.

The Binding Specificity of OppA Determines the Selectivity of the Oligopeptide ATP-binding Cassette Transporter

“…For the specificity studies, we used saturating OppA concentrations, implying that substrate binding or delivery to the translocator is not rate-determining. This mimics the in vivo situation where overexpression of OppA in wild-type L. lactis cells increased the amount of peptide binding but did not affect the rate of uptake (27). Furthermore, the K m for bradykinin transport in vivo (0.27 Ϯ 0.03 M) (27) and in vitro (0.9 Ϯ 0.2 M) are in the same range.…”

“…This mimics the in vivo situation where overexpression of OppA in wild-type L. lactis cells increased the amount of peptide binding but did not affect the rate of uptake (27). Furthermore, the K m for bradykinin transport in vivo (0.27 Ϯ 0.03 M) (27) and in vitro (0.9 Ϯ 0.2 M) are in the same range. Thus, both in vivo and in vitro there is a large excess of OppA over OppBCDF, and the rate-determining step for bradykinin uptake is in translocation and not in peptide binding.…”

Article

“…Le fait de surproduire OppA à la surface des cellules bactériennes [20] augmente la capacité d'OppA à lier un peptide donné mais n'augmente pas celle du transport sensu-stricto. L'étape limitante du transport d'un peptide n'est donc pas sa fixation sur OppA mais plus vraisemblablement son transfert vers le complexe transmembranaire OppBC (étapes III-IV du modèle Fig.…”

“…Pour savoir si la variabilité de séquen-ces des OppA peut être à l'origine de la variabilité de transport des peptides entre les souches, les gènes oppA des quatre souches (MG1363, IL1403, Wg2 et CNRZ437) ont été exprimés chez la souche MG1363 délétée pour le gène oppA (souche AMP15, [20]) [4].…”

Modelagem molecular das proteínas captadoras de molibdato (ModA) e oligopeptídeos (OppA) de Xanthomonas axonopodis pv. citri .

“…Xac mostra-se do tipo α/β, consistindo em uma mistura de folhas β rodeadas por α- A qualidade do modelo foi avaliada com o programa PROCHECK (LASKOWSKI et al, 1993 HILLES et al, 1986;FENNO et al, 2000;MONNET, 2003), Bsu (MONNET, 2003, Bbu (PICON et al, 2000) e Lla (TAME et al, 1995) …”