Flavin Binding to the High Affinity Riboflavin Transporter RibU

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B2) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655–5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (Kd for riboflavin is 0.6 nm), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure. The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B2) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655–5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (Kd for riboflavin is 0.6 nm), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure. Riboflavin (vitamin B2) is a water-soluble vitamin that is converted by flavokinases and FAD synthases to the cofactors FMN and FAD. These cofactors are indispensable for all living organisms, and they are involved in a wide range of reactions (1Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Vertebrates have lost the ability to synthesize riboflavin and the need to take up the vitamin from their gut (2Bowman B.B. Mccormick D.B. Rosenberg I.H. Annu. Rev. Nutr. 1989; 9: 187-199Crossref PubMed Scopus (26) Google Scholar). The proteins involved in epithelial uptake of riboflavin and membrane transport into other cell types in the body have not been identified yet. Most prokaryotes, fungi, and plants can synthesize riboflavin using pathways that have been well studied (3Bacher A. Eberhardt S. Fischer M. Kis K. Richter G. Annu. Rev. Nutr. 2000; 20: 153-167Crossref PubMed Scopus (223) Google Scholar). In addition, some prokaryotes and fungi can also take up riboflavin from the environment. The molecular identities of two very diverse types of riboflavin transporters have recently been established in yeast and bacteria (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google Scholar, 5Kreneva R.A. Gel'fand M.S. Mironov A.A. Yomantas Y.A. Kozlov Y.I. Mironov A.S. Perumov D.A. Russ. J. Genet. 2000; 36: 972-974Google Scholar, 6Reihl P. Stolz J. J. Biol. Chem. 2005; 280: 39809-39817Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A classical genetic approach revealed that riboflavin is transported across the plasma membrane of the yeast Saccharomyces cerevisiae by Mch5p, a homologue of mammalian monocarboxylate transporters (6Reihl P. Stolz J. J. Biol. Chem. 2005; 280: 39809-39817Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In vivo transport studies suggested that the protein facilitates diffusion and does not use metabolic energy for transport of riboflavin. In the bacteria Lactococcus lactis and Bacillus subtilis, two homologous riboflavin transporters, YpaA and RibU, respectively, were found (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google Scholar, 5Kreneva R.A. Gel'fand M.S. Mironov A.A. Yomantas Y.A. Kozlov Y.I. Mironov A.S. Perumov D.A. Russ. J. Genet. 2000; 36: 972-974Google Scholar). RibU is a 22.8-kDa membrane protein with five predicted membrane-spanning segments. RibU is not homologous to any previously characterized flavin-binding protein and belongs to a novel family of transport proteins with members found in Bacteria and Archaea. In vivo transport studies showed that RibU, like Mch5p in yeast, is likely to mediate facilitated diffusion (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google Scholar). Here we have overexpressed and purified RibU, and we have spectroscopically and biochemically characterized the binding of flavins to the transporter. Materials—Riboflavin, FMN, and FAD were obtained from Sigma, and roseoflavin was from Toronto Research Chemicals. The concentrations of the flavins in solution were determined photometrically (7Fischer M. Haase I. Feicht R. Richter G. Gerhardt S. Changeux J.P. Huber R. Bacher A. Eur. J. Biochem. 2002; 269: 519-526Crossref PubMed Scopus (34) Google Scholar). For roseoflavin an extinction coefficient of 31 mm-1·cm-1 at 505 nm was used (8Otto M.K. Jayaram M. Hamilton R.M. Delbruck M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 266-269Crossref PubMed Scopus (71) Google Scholar). Dodecyl-β-d-maltoside (DDM) 3The abbreviations used are: DDM, n-dodecyl-β-d-maltoside; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; ITC, isothermal titration calorimetry; RfBP, riboflavin-binding protein. was obtained from Anatrace. [3H]Riboflavin (24 Ci/mmol) was purchased from Moravek Biochemicals. All other chemicals were of analytical grade and obtained from commercial sources. Strains, Plasmids, and Growth Conditions—L. lactis strains NZ9000 (9Kuipers O.P. de Ruyter P.G.G.A. Kleerebezem M. de Vos W.M. J. Biotechnol. 1998; 64: 15-21Crossref Scopus (581) Google Scholar) and NZ9000-ΔribA (10Burgess C. O'Connell-Motherway M. Sybesma W. Hugenholtz J. van Sinderen D. Appl. Environ. Microbiol. 2004; 70: 5769-5777Crossref PubMed Scopus (175) Google Scholar) were used for cloning and expression. The ribU gene was engineered with the coding sequence for a C-terminal His10 tag by PCR. An NcoI site was introduced to coincide with the start codon, and an XbaI site was engineered immediately after the stop codon. These restriction sites were used to clone the ribU-His gene into expression plasmid pNZ8048 (9Kuipers O.P. de Ruyter P.G.G.A. Kleerebezem M. de Vos W.M. J. Biotechnol. 1998; 64: 15-21Crossref Scopus (581) Google Scholar) behind the nisinA promoter. The DNA sequence was checked (ServiceXS, The Netherlands). Three mutants (W68Y, W79Y, and W97Y) were made using the PCR overlap extension method and cloned into a pNZ8048-derived vector by ligase-independent cloning. 4E. R. Geertsma, manuscript submitted. The sequences coding for an N-terminal His tag and tobacco etch virus protease cleavage site were introduced. All mutations (TGG to TAT) were confirmed by DNA sequencing (Service XS, The Netherlands). L. lactis strains were grown at 30 °C in either GLX or M17 (Difco) medium supplemented with 1.0% (w/v) glucose and 5 μg/ml chloramphenicol. GLX contained 2% (w/v) gistex LS (Strik BV, Eemnes, The Netherlands) and 65 mm potassium phosphate (KPi), pH 7. Cells were grown in 1-liter bottles to an A600 of 0.7, followed by induction of the expression with 0.1% (v/v) culture supernatant of the nisin A producing strain NZ9700 (9Kuipers O.P. de Ruyter P.G.G.A. Kleerebezem M. de Vos W.M. J. Biotechnol. 1998; 64: 15-21Crossref Scopus (581) Google Scholar). The cells were harvested 1.5 h after induction. For the production of riboflavin-free RibU-His, 6 liters of cells were grown in M17 medium to an A600 of 0.7. Subsequently, cells were harvested, washed with sterile 50 mm KPi, pH 7, and transferred to a pH- and temperature-controlled fermenter (pH 6.8, 30 °C) containing 10 liters of chemically defined medium supplemented with 1.0% (w/v) glucose and 5 μg/ml chloramphenicol (11Molenaar D. Hagting A. Alkema H. Driessen A.J.M. Konings W.N. J. Bacteriol. 1993; 175: 5438-5444Crossref PubMed Google Scholar) without riboflavin. After 15 min, expression was induced with nisin A as described above. Cells were harvested after 1.5 h of induction and were frozen and stored at -80 °C. Purification of RibU-His—Membrane vesicles were prepared according to standard procedures (12van der Heide T. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7102-7106Crossref PubMed Scopus (150) Google Scholar) and stored at -80 °C. For solubilization, membrane vesicles were resuspended to a protein concentration of 1.5 mg/ml in buffer A (10% glycerol, 300 mm NaCl, 50 mm Tris/HCl, pH 8) supplemented with 15 mm imidazole/HCl, pH 8, and 0.5% DDM and incubated on ice for 30 min with occasional mixing by inverting. Unsolubilized material was spun down (20 min at 440,000 × g and 4 °C), and the supernatant was incubated with nickel-Sepharose chromatography medium equilibrated in buffer A containing 15 mm imidazole, pH 8 (1 ml of medium/15 mg of membrane protein; GE Heathcare), for 1 h at 4 °C with gentle agitation. The resin was into a and washed with of buffer A supplemented with mm imidazole, pH 8, and were in buffer A supplemented with mm imidazole, pH 8, and DDM in five of 5 mm pH was to the and the was a equilibrated with buffer (20 mm pH mm NaCl, and containing were and used were on a For of the purified protein the was and were prepared according to and M. Anal. Chem. 2000; PubMed Scopus Google Scholar). For riboflavin the was and protein of membrane vesicles was using the or the Anal. Biochem. PubMed Scopus Google Scholar) with as the The concentration of purified was determined by absorption at nm using an extinction coefficient of For of the extinction was The Netherlands). absorption were on a experiments were using an at °C. mg/ml in 50 mm Tris/HCl, pH or purified in was to the Riboflavin was in 50 mm Tris/HCl, pH or in buffer purified and into the were using the T. S. Anal. Biochem. 1989; PubMed Scopus Google Scholar). were on a fluorescence at °C in a For fluorescence titration purified was and of FMN, or FAD were in For FMN, and the and were and nm, in the of protein were as fluorescence was using an of was as described by G. Vos J. Poolman B. R.M. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) at °C in a mg/ml in 50 mm Tris/HCl, pH mm or purified in was to the riboflavin solution was into the in the of protein were as titration and were as described by G. Vos J. Poolman B. R.M. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) with the The or were from the of the and the were the concentration of was to the for and using 1 in is the concentration of binding sites in the is the substrate concentration is the is an is the concentration of of the concentration of binding sites by the titration of substrate was into as in in is the concentration of binding sites at the start of the and is the concentration of substrate in the In the of the were made to for the of substrate from the cell G. Vos J. Poolman B. R.M. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and Purification of riboflavin transporter RibU from L. lactis was with a C-terminal His10 tag and in L. lactis from the cells expressing the transporter were bright membrane vesicles from L. lactis are was from the with the DDM and purified using nickel-Sepharose and showed that the protein was more and the was mg of The of the purified transporter was confirmed by mass spectrometry of the like the membrane vesicles from the the purified protein was also The absorption spectrum in the of the purified protein had two with at and nm, respectively, and the had well defined at and 486 nm but The spectrum from the spectrum of riboflavin in the either the properties of riboflavin had changed when the bound to the transporter or was a yellow bound to We used mass spectrometry to the source of the yellow can used to membrane proteins directly in solution without the need for of as and buffer M. Anal. Chem. 2000; PubMed Scopus Google Scholar). A and mass of riboflavin in the and purified yellow RibU-His, respectively, in the range of riboflavin. The two very and have at of and to the riboflavin and riboflavin the riboflavin and riboflavin as previously by and Chem. Scopus Google Scholar). The at of also contained a of the a spectrum of is in The that riboflavin is bound to the purified other were found in the spectrum of RibU-His, and a of DDM not of the bound substrate to purified by mass mass of riboflavin with purified in with was on the and of Substrate-free the of riboflavin and of the a method to riboflavin-free RibU was to riboflavin from the protein and and a was to RibU at the expression We used a of L. lactis NZ9000 with a for (10Burgess C. O'Connell-Motherway M. Sybesma W. Hugenholtz J. van Sinderen D. Appl. Environ. Microbiol. 2004; 70: 5769-5777Crossref PubMed Scopus (175) Google an in the riboflavin (3Bacher A. Eberhardt S. Fischer M. Kis K. Richter G. Annu. Rev. Nutr. 2000; 20: 153-167Crossref PubMed Scopus (223) Google Scholar). riboflavin is for of L. the is completely on the uptake of riboflavin from the environment. The was grown to the in rich medium containing riboflavin. The RibU was not at but the of RibU was for the uptake of riboflavin from the medium. Cells were washed with and transferred to chemically defined medium from riboflavin had been The was in chemically defined and at the the expression of RibU was induced with nisin A. The cells to all riboflavin had been vesicles prepared from cells RibU at as membrane vesicles prepared from cells grown in rich medium but they were indicating that riboflavin-free had been The of bound riboflavin was confirmed by absorption of the purified protein and absorption of riboflavin were in the of of purified riboflavin-free of riboflavin to in a of the absorption at to nm and a of the at to were by a in the In addition, well defined appeared at 486 and the spectrum of riboflavin when bound to very to the spectrum of yellow purified from cells grown in rich medium not that riboflavin had been co-purified with the transporter. A titration was with an of riboflavin transport in L. lactis (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google Scholar). The absorption at 505 nm to nm, and the was by an fluorescence of riboflavin in the of concentrations of the of nm, of the riboflavin fluorescence was quenched when the was bound to The at nm that at a high was not by riboflavin fluorescence as was also in the buffer without protein and riboflavin The tryptophan fluorescence spectrum of had a at nm and the fluorescence was quenched by the binding of riboflavin and roseoflavin for concentrations of riboflavin and roseoflavin quenched and of the tryptophan respectively, and in a of the of were used to the and the binding stoichiometry of and isothermal titration T. S. Anal. Biochem. 1989; PubMed Scopus Google fluorescence titration (7Fischer M. Haase I. Feicht R. Richter G. Gerhardt S. Changeux J.P. Huber R. Bacher A. Eur. J. Biochem. 2002; 269: 519-526Crossref PubMed Scopus (34) Google and G. Vos J. Poolman B. R.M. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). For the of the binding an of the protein concentration was We used and an of was determined by 5 the results of the of purified RibU-His, from an of nm was and a binding stoichiometry of of riboflavin protein the that a of the purified protein and in the of the protein concentration for we that is one riboflavin-binding site protein using riboflavin very concentrations of protein and the affinity of for riboflavin is very the of the was not very is a more the used of protein concentrations as as nm, a more for the of 0.6 nm 6 and and FMN also bound with high affinity to the but FAD did not is with in vivo transport in was found that roseoflavin and FMN uptake of riboflavin by L. lactis cells and that are likely to transported into the cells by RibU (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google and binding of and abbreviations used are as fluorescence titration nm, fluorescence titration nm, purified protein; indicate membrane vesicles expressing stoichiometry The abbreviations used are as fluorescence titration nm, fluorescence titration nm, purified protein; indicate membrane vesicles expressing in a titration of with riboflavin. fluorescence of riboflavin when to the protein solution or to buffer The fluorescence the two was and the riboflavin concentration the and binding stoichiometry were as described was also used to the binding in membrane vesicles expressing riboflavin-free The in membrane vesicles were to the determined in In the membrane vesicles of binding sites for riboflavin were found mg of membrane protein. is one binding site for riboflavin RibU the expression of RibU in the membrane vesicles was more of membrane protein. is with of membrane vesicles expressing RibU, showed of RibU. Mutagenesis of tryptophan and tryptophan was to and the riboflavin were determined by fluorescence titration the of the tryptophan residues by riboflavin binding was in the The riboflavin of the and mutants (Kd of nm were very to the indicating that and are not involved in substrate binding. In the riboflavin of the had changed dramatically (Kd of In to the protein the tryptophan fluorescence of the was quenched to by riboflavin binding we that the tryptophan fluorescence of the protein with riboflavin can to The also that the fluorescence of and was completely quenched upon riboflavin binding. the fluorescence of the was quenched to a by riboflavin binding the protein the of tryptophan fluorescence and the of were very to the protein not indicating that tryptophan to the tryptophan fluorescence of RibU. of riboflavin to the transporter RibU results in in the absorption spectrum of the The of flavins bound to proteins on the properties of the binding pocket. The spectrum of riboflavin bound to RibU very well resolved absorption bands at 441, 464, and 486 nm, indicating a of the S. Biochem. J. PubMed Scopus Google Scholar, R.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Google Scholar). the of the absorption from to nm and the of the at nm are of a hydrophobic binding pocket Mccormick D.B. Biochem. PubMed Scopus Google Scholar, M. J. Biochem. PubMed Scopus Google Scholar). The are of the that when riboflavin to RfBP, a soluble protein for the vitamin in Mccormick D.B. Biochem. PubMed Scopus Google Scholar, M. J. Biochem. PubMed Scopus Google Scholar, Annu. Rev. Nutr. PubMed Scopus Google Scholar). The of been J. PubMed Scopus Google the are not in the In the the binding pocket of riboflavin is hydrophobic with the of riboflavin stacked the of the of a tryptophan and a of riboflavin to results in an almost of the riboflavin is to the of the and the In addition, the tryptophan fluorescence of is quenched by when riboflavin or riboflavin are bound Mccormick D.B. Biochem. PubMed Scopus Google Scholar, M. J. Biochem. PubMed Scopus Google been to the of 5 the 6 in in the of riboflavin J. PubMed Scopus Google Scholar). of riboflavin with residues and of the riboflavin fluorescence have also been in other riboflavin-binding proteins in and B. D. Full Text Full Text PDF PubMed Scopus (71) Google Scholar, S. Haase I. S. M. Bacher A. Huber R. Fischer M. J. Biol. 2002; PubMed Scopus Google of riboflavin to RibU also in of riboflavin and tryptophan fluorescence to as for the riboflavin of and RibU are very and 0.6 nm, We that the riboflavin-binding sites in RibU and and that binding are very hydrophobic as by the of the absorption that sequence and RibU. The of the tryptophan fluorescence of the that the are in a when riboflavin is not bound. RibU tryptophan and at the C-terminal of membrane-spanning and and at the N-terminal of of is completely in the family of proteins (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google but Trp-68 and are in of well and we that their fluorescence quenched by riboflavin. Mutagenesis of of the tryptophan residues to revealed that and are not involved in riboflavin but the properties of the changed dramatically with the protein. The changed to is likely that that tryptophan is stacked with in the binding pocket of RibU, the tryptophan fluorescence of Trp-68 was quenched to by binding of the The affinity of for riboflavin is very high (Kd 0.6 nm), transporters with the known affinity for their For a substrate affinity that is 6 of M. J. 2000; PubMed Scopus Google Scholar). affinity binding to RibU is to riboflavin from the in is at concentration (2Bowman B.B. Mccormick D.B. Rosenberg I.H. Annu. Rev. Nutr. 1989; 9: 187-199Crossref PubMed Scopus (26) Google Scholar). The affinity of RibU for its substrate is with the riboflavin binding affinity (Kd in membrane vesicles of B. G. M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). likely the binding of riboflavin to B. is by a homologue of RibU R.A. Gel'fand M.S. Mironov A.A. Yomantas Y.A. Kozlov Y.I. Mironov A.S. Perumov D.A. Russ. J. Genet. 2000; 36: 972-974Google Scholar). The high binding affinity of RibU and riboflavin a for the of riboflavin in the and the concentration of riboflavin to is very likely by metabolic of riboflavin by synthases that the vitamin to FMN and FAD G. M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the high affinity for in transport in membrane vesicles or in with the protein. the transporter facilitated diffusion (4Burgess C.M. Slotboom D.J. Geertsma E.R. Duurkens R.H. Poolman B. van Sinderen D. J. Bacteriol. 2006; 188: 2752-2760Crossref PubMed Scopus (67) Google of substrate in the in the of metabolic and transport is by binding. have been for with the high affinity transporter T. Microbiol. 2000; PubMed Scopus Google Scholar). We have used mass spectrometry to that the substrate riboflavin is co-purified with mass spectrometry is used to the of purified membrane proteins M. Anal. Chem. 2000; PubMed Scopus Google Scholar). The of other ionization in the of membrane proteins is that the to the membrane protein not have to completely M. Anal. Chem. 2000; PubMed Scopus Google and the is the first in the been used to the substrate bound to a membrane transport protein. of the of membrane transport proteins is as have revealed of transporters with known mass spectrometry a to bound to purified membrane proteins. The that the substrate is co-purified with the transporter for high affinity binding as RibU, but proteins that bind their with affinity the substrate the have that of co-purified with proteins of their binding that the protein concentrations are high the the G. of the from of Scholar). of membrane proteins of high concentration are purified for and mass spectrometry a for substrate identification in structural in membrane proteins of are We Poolman for of the manuscript and for with the mass for on for on for for of the plasmid and van Sinderen of for the of NZ9000

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