PnuT uses a facilitated diffusion mechanism for thiamine uptake.

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Bacterial pyridine nucleotide uptake (Pnu) family transporters facilitate B-type vitamin uptake.
• PnuT transporters specifically transport vitamin B1 (thiamine).
• Previous hypotheses suggested Pnu transporters act as facilitators for passive vitamin transport, followed by cytoplasmic metabolic trapping.

Purpose of the Study:

• To elucidate the transport mechanism of PnuT transporters.
• To determine the substrate specificity and transport properties of PnuT_Sw.
• To identify key residues involved in thiamine transport and gating.

Main Methods:

• Purification of PnuT_Sw from *Shewanella woodyi*.
• Reconstitution of purified PnuT_Sw into liposomes.
• Transport assays and substrate binding studies.
• Site-directed mutagenesis guided by the PnuC crystal structure.

Main Results:

• Thiamine transport direction is solely determined by the electrochemical gradient, indicating facilitated diffusion.
• PnuT_Sw transports thiamine and its analogues (pyrithiamine, oxythiamine) but not phosphorylated derivatives (TMP, TPP).
• Mutagenesis identified residues critical for substrate binding and transporter gating.

Conclusions:

• PnuT_Sw utilizes a facilitated diffusion mechanism for thiamine uptake.
• The transporter's specificity for non-phosphorylated thiamine supports a metabolic trapping model.
• This mechanism contrasts with active transport systems found in other bacterial thiamine transporters.