title: EF0176 and EF0177 from Enterococcus faecalis V583 are substrate-binding lipoproteins involved in ABC transporter mediated ribonucleoside uptake.
abstract: One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
authors: Ingrid Lea
authors: IL
authors: Karlskås
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
authors: Zhian
authors: Z
authors: Saleihan
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
authors: Helge
authors: H
authors: Holo
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
authors: Geir
authors: G
authors: Mathiesen
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway Vincent.eijsink@nmbu.no.
authors: Vincent G H
authors: VG
authors: Eijsink
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D019295:Computational Biology; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D008074:Lipoproteins / Q000378:metabolism*; D005810:Multigene Family; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008074:Lipoproteins