Biochemical characterization of ThiT from Lactococcus lactis: a thiamin transporter with picomolar substrate binding affinity.
The putative thiamin transporter ThiT from Lactococcus lactis was overproduced in the membrane of lactococcal cells. In vivo transport assays using radiolabeled thiamin demonstrated that ThiT indeed was involved in thiamin transport. The protein was solubilized from the membranes and purified in detergent solution. Size exclusion chromatography coupled to static light scattering, refractive index, and UV absorbance measurements (SEC-MALLS) showed that ThiT is a monomer of 22.7 kDa in detergent solution. When the cells overexpressing ThiT had been cultivated in complex growth medium, all binding sites of the purified protein were occupied with substrate, which had copurified with the protein. MALDI-TOF mass spectrometry analysis confirmed that the copurified substance was thiamin. Substrate-depleted ThiT was obtained by expressing the protein in cells that were cultivated in chemically defined growth medium without thiamin. The intrinsic tryptophan fluorescence of substrate-depleted ThiT was strongly quenched upon thiamin binding. The quenching of the fluorescence was used to determine dissociation constants for thiamin and related compounds. ThiT had an unusually high affinity for thiamin (K(D) = 122 +/- 13 pM) and bound the substrate with a 1:1 (protein:ligand) stoichiometry. TPP, TMP, and pyrithiamin bound to ThiT with nanomolar affinity. A multiple sequence alignment of ThiT homologues revealed that well-conserved residues were clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanning segments 5 and 6. Mutational analysis of the conserved residues in this region combined with binding assays of thiamin and related compounds was used to build a model of the high-affinity binding site. The model was compared with thiamin binding sites of other proteins and interpreted in terms of the transport mechanism.
Erkens
Guus B
GB
Department of Biochemistry, University of Groningen, Groningen Biomolecular Science and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Slotboom
Dirk Jan
DJ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D006801:Humans; D013294:Lactococcus lactis / Q000378:metabolism*; D011485:Protein Binding; D013831:Thiamine / Q000031:analogs & derivatives / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D013831:Thiamine