Abstract

Substrate Specificity of the Citrate Transporter CitP of Lactococcus lactis Agata M. Pudlik a , b , c and Juke S. Lolkema b a Top Institute Food and Nutrition, Wageningen, The Netherlands b Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands c The Kluyver Centre for Genomics of Industrial Fermentations/NCSB, Delft, The Netherlands ABSTRACT The citrate transporter CitP of lactic acid bacteria catalyzes electrogenic precursor-product exchange of citrate versus l -lactate during citrate-glucose cometabolism. In the absence of sugar, l -lactate is replaced by the metabolic intermediates/end products pyruvate, α-acetolactate, and acetate. In this study, the binding and translocation properties of CitP were analyzed systematically for a wide variety of mono- and dicarboxylates of the form X-CR 2 -COO − , where X represents OH (2-hydroxy acid), O (2-keto acid), or H (acid) and R groups differ in size, hydrophobicity, and composition. It follows that CitP is a very promiscuous carboxylate transporter. A carboxylate group is both essential and sufficient for recognition by the transporter. A C-2 atom is not essential, formate is a substrate, and C-2 may be part of a ring structure, as in benzoate. The R group may be as bulky as an indole ring structure. For all monocarboxylates of the form X-CHR-COO − , the hydroxy (X = OH) analogs were the preferred substrates. The preference for keto (X = O) or acid (X = H) analogs was dependent on the bulkiness of the R group, such that the acid was preferred for small R groups and the 2-ketoacid was preferred for more bulky R groups. The C 4 to C 6 dicarboxylates succinate, glutarate, and adipate were also substrates of CitP. The broad substrate specificity is discussed in the context of a model of the binding site of CitP. Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone.