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 catal yze citrate-lactate and malate-lactate exchange, respectively. Both transpo rters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR2-COO- [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-1 8146]. 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 moti f drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP wer e 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 spec ific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in b inding when the R groups are hydrophobic and as large as a benzyl group. Ho wever, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates wi th large hydrophobic R groups. These differences between CitP and MleP expl ain why citrate is a substrate of CitP and not of MleP. The results are dis cussed in the context of a model for the interaction between sites on the p rotein and functional groups on the substrates in the binding pockets of th e two proteins.