The conserved C-terninus of the citrate (CitP) and malate (MleP) transporters of lactic acid bacteria is involved in substrate recognition

The membrane potential-generating transporters CitP of Leuconostoc mesenter oides and MleP of Lactococcus lactis are homologous proteins with 48% ident ical residues that catalyze citrate-lactate and malate-lactate exchange, re spectively. The two transporters are highly specific for substrates contain ing a 2-hydroxycarboxylate motif (HO-CR2-COO-) in which substitutions of th e R groups are tolerated well. Differences in substrate specificity between MleP and CitP are based on subtle changes in the interaction of the protei n with the R groups affecting both binding and translocation properties. Th e conserved, 46-residue long C-terminal region of the transporters containi ng the C-terminal putative transmembrane segment XI was investigated for it s role in substrate recognition by constructing chimeric transporters. Repl acement of the C-terminal region of MleP with that of CitP and vice versa d id not alter the exchange kinetics with the substrates malate and citrate, indicating that the main interactions between the proteins and di- and tric arboxylate substrates were not altered. In contrast, the interaction of the proteins with the monocarboxylate substrates mandelate and 2-hydroxyisoval erate changed in a complementary manner. The affinity of CitP for the S-ena ntiomers of these substrates was at least 1 order of magnitude lower than o bserved for MleP, Introduction of the C-terminal residues of MleP in CitP r esulted in a higher affinity and vice versa. Interchanging the C-termini ha d a more complicated effect on the R-enantiomers, affecting different kinet ic parameters with different substrates, indicating multiple interactions o f the R groups at this side of the binding pocket. It is suggested that the binding pocket is located between transmembrane segment XI and the other t ransmembrane segments of the transporters.