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DECARBOXYLATE Résumé de rapport

Project ID: QLK1-CT-2002-02388
Financé au titre de: FP5-LIFE QUALITY
Pays: Netherlands

Binding site model for di- and tricarboxylate transporters

Biogenic amines (BA) are the product of decarboxylation pathways in food bacteria and a major cause of food poisoning. Substrates of decarboxylation pathways are amino acids that are converted into the corresponding amines or amino acids (e.g. histidine/histamine, aspartate/alanine, tyrosine/tyramine), but also di/tricarboxylates that are converted into monocarboxylates (e.g. malate/lactate, citrate/lactate). The latter are beneficial in fermentation processes. The pathways consist of a transporter that catalyses the translocation of the substrate into the cell coupled to the secretion of the metabolic end product of the same substrate out of the cell, and a decarboxylase. In most cases the end product is the direct decarboxylation product of the substrate and, therefore, both are structurally related. The transporters are at the gate of the pathways and any unwanted activity is best attacked there. We aim to develop inhibitors of BA production for food preservatives, and to engineer the transporters to prevent inhibition by naturally occurring compounds of the beneficial citrate and malate pathways. Both goals would benefit from a detailed structural model of the binding site of the transporter.

The principle of binding both substrate and end-product was studied by determining the global structure of the transporters and by identifying and characterising the binding site of the citrate and malate transporters MleP and CitP. These studies involve the membrane topology, domain structure and the residues on the protein that co-ordinate the different groups of the substrates. The studies made use of bioinformatics approaches in analyzing the gene families to which the transporters belong and of experimental approaches to identify residues in the binding site and membrane topologies.

The citrate and malate transporters are members of the 2-hydroxycarboxylate transporter (2HCT) family. The structural model of the proteins of the 2HCT family shows two homologous domains consisting of 5 transmembrane segments (TMS) each, and separated by a large hydrophilic loop that resides in the cytoplasm. An additional TMS is present at the N-terminus of the protein. This domain is not part of the two-domain structure. A major consequence of the odd number of TMSs in the two domains is that they have opposite orientations in the membrane. The N-terminus of the N-terminal domain is in the periplasm, while the N-terminus of the C-terminal domain is in the cytoplasm. Homologous domains with inverted topologies turn out to be a common structural motif in membrane proteins. The corresponding loops between the fourth and fifth TMS in each domain fold into pore-loop structures. Both regions contain an extraordinarily high fraction of residues with small side chains, which may reflect a compact packing of the loops between the transmembrane segments. A sequence motif GGxG is located at the centre of the pore-loop regions and may represent the vertex of the loops. The loop between TMS VIII and IX in the C-terminal domain folds into an amphipathic surface helix. This feature is not observed in the N-terminal domain.

The pore-loop structures in the two domains enter the protein from opposite sites of the membrane and are likely to contact each other in the three dimensional structure where they form the substrate binding site and the translocation site. Close to this structural arrangement, an arginine residues in TMS XI in the C-terminal domain is highly conserved in the family of proteins. The residue is interacting with one of the carboxylate groups on the substrates and places the substrate binding site at the cytoplasm/membrane interface. Interaction between a second carboxylate group and the hydroxyl group on the substrates are essential for binding, but the interacting residues on the proteins are still elusive.

Informations connexes


Julius LOLKEMA, (Group leader)
Tél.: +31-50-3632155
Fax: +31-50-3632154