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Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

ABSTRACT: The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

SUBMITTER: Soysa R

PROVIDER: S-EPMC3952013 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

Soysa Radika R Venselaar Hanka H Poston Jacqueline J Ullman Buddy B Hasne Marie-Pierre MP

The Biochemical journal 20130601 3

The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of Tc ...[more]

PMID: 23535070

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Project description:Lactic acid bacteria (LAB) play a key role in many food fermentations. However, some LAB species can also cause food spoilage, e.g., through the formation of biogenic amines. Paucilactobacillus wasatchensis is a LAB that causes late gas production in Cheddar cheese, the molecular causes of which are not fully understood. This study reports on the ability of P. wasatchensis WDC04 to produce cadaverine and putrescine in broth supplemented with lysine and ornithine, as well as in a model cheese. The raclette-type semi-hard cheese produced with P. wasatchensis as an adjunct culture contained 1,085 mg kg-1 of cadaverine and 304 mg kg-1 of putrescine after 120 days of ripening. We identified two ornithine decarboxylase genes (odc) and a putrescine-ornithine antiporter gene (potE) in the genome sequence of P. wasatchensis. We could show that the two odc genes, which are located on two contigs, are contiguous and form the genetic cluster odc2-odc1-potE. Alignment searches showed that similar gene clusters exist in the genomes of Levilactobacillus paucivorans DSMZ22467, Lentilactobacillus kribbianus YH-lac9, Levilactobacillus hunanensis 151-2B, and Levilactobacillus lindianensis 220-4. More amino acid sequence comparisons showed that Odc1 and Odc2 shared 72 and 69% identity with a lysine and ornithine decarboxylase from Ligilactobacillus saerimneri 30a, respectively. To clarify the catalytic activities of both enzymes, the odc-coding genes were cloned and heterologously expressed as His-tagged fusion protein. The purified Odc1 protein decarboxylated lysine into cadaverine, while the recombinant Odc2 protein preferentially produced putrescine from ornithine but also exhibited low lysine decarboxylating activity. Both enzymes were active at pH of 5.5, a value often found in cheese. To our knowledge, this is only the second lysine decarboxylase in LAB whose function has been verified. The tandem arrangement of the genes in a single cluster suggests a gene duplication, evolving the ability to metabolize more amino. Divergent substrate preferences highlight the necessity of verifying the functions of genes, in addition to automatic annotation based on sequence similarity. Acquiring new biochemical data allows better predictive models and, in this case, more accurate biogenic amine production potential for LAB strains and microbiomes.

Dataset Information

Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

Publications

Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

Similar Datasets

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