Stereoselectivity of the membrane potential-generating citrate and malate transporters of lactic acid bacteria.
The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR(2)-COO(-) [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO(-) groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.
Bandell
M
M
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Lolkema
J S
JS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D001665:Binding Sites; D001692:Biological Transport; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D002384:Catalysis; D007700:Kinetics; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism*; D007934:Leuconostoc / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008293:Malates / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D027361:Organic Anion Transporters; D013237:Stereoisomerism; D027981:Symporters
D001426:Bacterial Proteins; D002264:Carboxylic Acids; D002352:Carrier Proteins; C096373:CitP protein, Lactobacillales; D008051:Lipid Bilayers; D008293:Malates; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D027981:Symporters; C098940:malate permease protein, Bacteria; D019344:Lactic Acid; C030298:malic acid