PnuT uses a facilitated diffusion mechanism for thiamine uptake
| Autor: | Rajkumar Singh, Alisa A. Garaeva, Dirk Jan Slotboom, Michael Jaehme, Ria H. Duurkens |
|---|---|
| Přispěvatelé: | Enzymology |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Shewanella Passive transport Physiology 030106 microbiology ECF TRANSPORTER Diffusion 03 medical and health sciences chemistry.chemical_compound ABC TRANSPORTERS Bacterial Proteins BINDING CRYSTAL-STRUCTURE BIOSYNTHESIS Thiamine Electrochemical gradient Research Articles Binding Sites Facilitated diffusion IDENTIFICATION Thiamine transport MEMBRANE-PROTEINS COUPLING FACTOR TRANSPORTER Membrane Transport Proteins food and beverages Thiamine monophosphate PROKARYOTES 030104 developmental biology chemistry BACTERIA Biophysics Pyrithiamine human activities Thiamine pyrophosphate Protein Binding Research Article |
| Zdroj: | Journal of general physiology, 150(1):jgp.201711850, 41-50 The Journal of General Physiology |
| ISSN: | 0022-1295 |
| Popis: | The bacterial pyridine nucleotide uptake family of transporters mediates the uptake of B-type vitamins, but their transport mechanism is unknown. Jaehme et al. show that the PnuT thiamine transporter utilizes a facilitated diffusion mechanism and supports metabolic trapping of phosphorylated thiamine. Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters. |
| Databáze: | OpenAIRE |
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