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• W2065291275 abstract "Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr73, Tyr89, Tyr90, Glu204, Tyr235, Asp303, and Tyr423 were strongly involved in both uptake and excretion and that Tyr55, Glu76, Tyr246, Tyr310, Cys370, and Glu377 were moderately involved in both activities. Mutations of Trp43, Tyr57, Tyr107, Tyr366, and Tyr368 mainly affected uptake activity, and Trp41, Tyr174, Asp185, and Glu408 had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the Km value. Mutation of Arg299 mainly affected excretion, suggesting that Arg299 is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys370 seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys125, Cys389, or Cys394 together with Cys370. The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys370. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII. Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr73, Tyr89, Tyr90, Glu204, Tyr235, Asp303, and Tyr423 were strongly involved in both uptake and excretion and that Tyr55, Glu76, Tyr246, Tyr310, Cys370, and Glu377 were moderately involved in both activities. Mutations of Trp43, Tyr57, Tyr107, Tyr366, and Tyr368 mainly affected uptake activity, and Trp41, Tyr174, Asp185, and Glu408 had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the Km value. Mutation of Arg299 mainly affected excretion, suggesting that Arg299 is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys370 seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys125, Cys389, or Cys394 together with Cys370. The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys370. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII. Polyamines (putrescine, spermidine, and spermine) play important roles in cell proliferation and differentiation (1Cohen S.S. A Guide to the Polyamines. Oxford University Press, New York1998: 1-543Google Scholar, 2Igarashi K. Kashiwagi K. Biochem. Biophys. Res. Commun. 2000; 271: 559-564Crossref PubMed Scopus (718) Google Scholar, 3Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (764) Google Scholar), and cellular polyamine content is regulated by biosynthesis, degradation, and transport (4Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar, 5Igarashi K. Kashiwagi K. Biochem. J. 1999; 344: 633-642Crossref PubMed Scopus (222) Google Scholar, 6Igarashi K. Ito K. Kashiwagi K. Res. Microbiol. 2001; 152: 271-278Crossref PubMed Scopus (79) Google Scholar). With regard to transport, the properties of three polyamine transport systems were characterized in Escherichia coli (7Vassylyev D.G. Tomitori H. Kashiwagi K. Morikawa K. Igarashi K. J. Biol. Chem. 1998; 273: 17604-17609Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 8Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). They include spermidine-preferential and putrescine-specific uptake systems, which belong to the family of ATP-binding cassette transporters, and a protein, PotE, involved in the excretion of putrescine by a putrescine-ornithine antiporter activity. Furthermore, it has been reported that cadaverine and aminopropylcadaverine function as compensatory polyamines for cell growth (10Igarashi K. Kashiwagi K. Hamasaki H. Miura A. Kakegawa T. Hirose S. Matsuzaki S. J. Bacteriol. 1986; 166: 128-134Crossref PubMed Scopus (155) Google Scholar), and CadB, a cadaverine-lysine antiporter, is strongly involved in cell growth at acidic pH like PotE (11Neely M.N. Olson E.R. J. Bacteriol. 1996; 178: 5522-5528Crossref PubMed Google Scholar, 12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar). Analogous to the speF-potE operon (13Kashiwagi K. Suzuki T. Suzuki F. Furuchi T. Kobayashi H. Igarashi K. J. Biol. Chem. 1991; 266: 20922-20927Abstract Full Text PDF PubMed Google Scholar), cadB is one component of the cadBA operon, in which cadA encodes lysine decarboxylase (14Meng S.Y. Bennett G.N. J. Bacteriol. 1992; 174: 2659-2669Crossref PubMed Google Scholar, 15Watson N. Dunyak D.S. Rosey E.L. Slonczewski J.L. Olson E.R. J. Bacteriol. 1992; 174: 530-540Crossref PubMed Google Scholar). Expression of the cadBA operon is regulated by a positive regulator CadC, which is encoded by adjacent cadC gene (14Meng S.Y. Bennett G.N. J. Bacteriol. 1992; 174: 2659-2669Crossref PubMed Google Scholar, 15Watson N. Dunyak D.S. Rosey E.L. Slonczewski J.L. Olson E.R. J. Bacteriol. 1992; 174: 530-540Crossref PubMed Google Scholar), and is induced by acidic pH and lysine (11Neely M.N. Olson E.R. J. Bacteriol. 1996; 178: 5522-5528Crossref PubMed Google Scholar). The cadBA and speF-potE operons contribute to an increase in pH of the extracellular medium through excretion of cadaverine and putrescine, the consumption of a proton, and a supply of carbon dioxide during the decarboxylation reaction (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar, 16Takayama M. Ohyama T. Igarashi K. Kobayashi H. Mol. Microbiol. 1994; 11: 913-918Crossref PubMed Scopus (33) Google Scholar) so that expression of these two operons is important for cell growth at acidic pH. It is also shown that expression of the speF-potE operon is increased when the cadBA operon is not induced (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar). Other antiport systems for basic amino acids and their amine products (histidine/histamine and arginine/agmatine), coupled with decarboxylation, have been reported to generate a proton motive force in Lactobacillus buchneri and E. coli (17Molenaar D. Bosscher J.S. ten Brink B. Driessen A.J.M. Konings W.N. J. Bacteriol. 1993; 175: 2864-2870Crossref PubMed Google Scholar, 18Gong S. Richard H. Foster J.W. J. Bacteriol. 2003; 185: 4402-4409Crossref PubMed Scopus (99) Google Scholar). At neutral pH, both CadB and PotE have cadaverine and putrescine uptake activity and contribute to cell growth of a polyamine-requiring mutant MA261 (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar, 13Kashiwagi K. Suzuki T. Suzuki F. Furuchi T. Kobayashi H. Igarashi K. J. Biol. Chem. 1991; 266: 20922-20927Abstract Full Text PDF PubMed Google Scholar). Although we have recently studied the function of CadB in detail (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar), the structure of CadB is not well understood. When homology between the amino acid sequences of CadB and PotE was compared, high sequence similarity was observed (30.7% overall identity) (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar). We have previously identified amino acid residues involved in putrescine uptake and excretion by PotE (8Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this study, we have identified a likely cadaverine recognition site on CadB based on the information concerning putrescine recognition by PotE.EXPERIMENTAL PROCEDURESPlasmid and Site-directed Mutagenesis of cadB Gene—Plasmid pMWcadB was prepared using pMW119 (Nippon Gene Co.) as described previously (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar). Site-directed mutagenesis of cadB gene was carried out with the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing using a Seq 4 × 4 personal sequencing system (Amersham Biosciences) or CEQ8000 DNA genetic analysis system (Beckman Coulter). A list of oligonucleotide primers for mutagenesis has not been included but is available from the authors upon request. pUCcadB was constructed by inserting 1.5 kbp BamHI and EcoRI fragments of pMWcadB into the same restriction sites of pUC119 (Takara Shuzo Co.).Culture Conditions—E. coli JM109 (recA1 supE44 endA1 gyrA96 relA1 thi Δ (lac-proAB)/F′ (traD36 proAB+ lacIq lacZΔM15)) (19Sambrook J. Fritsch E.F. Maniatis T. Sambrook J. Russell D.W. 3rd Ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: A3.6-A3.10Google Scholar) containing pMWcadB was cultured in a 19-amino acid supplemented medium containing 0.4% glucose for uptake by intact cells or 1% glycerol for preparation of inside-out membrane vesicles (20Kashiwagi K. Igarashi K. J. Bacteriol. 1988; 170: 3131-3135Crossref PubMed Google Scholar). Ampicillin (100 μg/ml) and/or kanamycin (50 μg/ml) were added to the medium, if necessary. Where indicated, 0.2 mm 2-mercaptoethanol was added to the medium.Cadaverine Uptake by Intact Cells—E. coli JM109/pMW119 or JM109/pMWcadB cultured until A540 = 0.5 in the presence of 0.5 mm isopropyl β-d-thiogalactopyranoside (IPTG) 2The abbreviations used are: IPTG, isopropyl β-d-thiogalactopyranoside; NEM, N-ethylmaleimide. was suspended in buffer I containing 0.4% glucose, 62 mm potassium phosphate, pH 7.0, 1.7 mm sodium citrate, 7.6 mm (NH4)2SO4, and 0.41 mm MgSO4 to yield a protein concentration of 0.1 mg/ml. The cell suspension (0.48 ml) was preincubated at 30 °C for 5 min, and the reaction was started by the addition of 0.02 ml of 0.25 mm [14C]cadaverine (370 MBq/mmol, Sigma). Thus, the concentration of [14C]cadaverine in the reaction mixture was 10 μm. After incubation at 30 °C for 30 s to 3 min, the cells were collected on cellulose acetate filters (0.45 μm; Advantec Toyo Co.), and the filters were washed three times with a total of 9 ml of buffer I. To determine the Km values of cadaverine for cadaverine uptake activity of cadB mutants, 10-200 μm cadaverine was used as a substrate. The radioactivity on the filters was measured with a liquid scintillation spectrometer. Protein content was determined by the method of Lowry et al. (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).Preparation of Inside-out Membrane Vesicles and Cadaverine Uptake by the Vesicles—Cadaverine-lysine antiport activity by CadB was estimated by measuring cadaverine uptake by inside-out membrane vesicles prepared according to the method of Houng et al. (22Houng H.S. Lynn A.R. Rosen B.P. J. Bacteriol. 1986; 168: 1040-1044Crossref PubMed Google Scholar). E. coli JM109/pMWcadB cells were cultured until A540 = 0.5 in the presence of 0.5 mm IPTG. Inside-out membrane vesicles were prepared by French press treatment (10,000 p.s.i.) of the cells suspended in a buffer containing 100 mm potassium phosphate buffer, pH 6.6, 10 mm EDTA in the presence of 2.5 mm lysine (23Kashiwagi K. Miyamoto S. Suzuki F. Kobayashi H. Igarashi K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4529-4533Crossref PubMed Scopus (115) Google Scholar). After removal of unbroken cells and cell debris, membrane vesicles were collected by ultracentrifugation (170,000 × g, 1 h). Membrane vesicles were washed with a buffer containing 10 mm Tris-HCl, pH 7.5, 0.14 m KCl, 2 mm 2-mercaptoethanol, and 10% glycerol, collected by ultracentrifugation, and suspended in the same buffer at the protein concentration of 5-10 mg/ml. The reaction mixture (0.1 ml) for the uptake by inside-out membrane vesicles contained 10 mm Tris-HCl, pH 8.0, 0.14 m KCl, 100 μm [14C]cadaverine (1.48 GBq/mmol), and 100 μg of inside-out membrane vesicle protein. After incubation at 22 °C for 10 s to 1 min, membrane vesicles were collected on cellulose nitrate filters (0.45 μm; Advantec Toyo Co.) and washed twice with a total of 9 ml of washing buffer (10 mm Tris-HCl, pH 8.0, and 0.14 m KCl). To determine the Km values of cadaverine for cadaverine-lysine antiport activity of cadB mutants, 100-2000 μm cadaverine was used as a substrate. Radioactivity on the filters was measured with a liquid scintillation spectrometer.Western Blot Analysis of CadB Protein—Rabbit antibody for the CadB protein was prepared according to the method of Posnett et al. (24Posnett D.N. McGrath H. Tam J.P. J. Biol. Chem. 1988; 263: 1719-1725Abstract Full Text PDF PubMed Google Scholar) using the multiple antigenic peptide, KVY-GEVDSNGIPKK, which correspond to amino acids 308-321 of the CadB protein. For Western blot analysis of CadB, inside-out membrane vesicles (30 μg protein) were separated by SDS-poly-acrylamide gel electrophoresis on a 12% acrylamide gel and transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore). CadB protein was detected with ProtoBlot Western blot AP System (Promega), except that 0.2% Triton X-100 was used instead of 0.05% Tween 20 (25Nielsen P.J. Manchester K.L. Towbin H. Gordon J. Thomas G. J. Biol. Chem. 1982; 257: 12316-12321Abstract Full Text PDF PubMed Google Scholar).Reactivity of [14C]NEM ([ethyl-1-14C]N-Ethylmaleimide) with CadB Protein—E. coli JM109/pUCcadB cells were cultured until A540 = 0.5 in a 19-amino acid supplemented medium containing 1% glycerol and 0.5 mm IPTG with or without 0.2 mm 2-mecaptoethanol. Right-side-out membrane vesicles were prepared as described previously (26Kashiwagi K. Kobayashi H. Igarashi K. J. Bacteriol. 1986; 165: 972-977Crossref PubMed Google Scholar). Reactivity of [14C]NEM with CadB was tested according to the method of Sahin-Tóth et al. (27Sahin-Tóth M. Gunawan P. Lawrence M.C. Toyokuni T. Kaback H.R. Biochemistry. 2002; 41: 13039-13045Crossref PubMed Scopus (28) Google Scholar). Right-side-out membrane vesicles (200 μg protein) were suspended with 50 mm sodium phosphate, pH 7.5, in a final volume of 50 μl, and 10 μl of 2.4 mm [14C]NEM (2.1 GBq/mmol, American Radiolabeled Chemicals Inc.) was added. Incubation was carried out at 22 °C for 10 min. Reactions were terminated by adding 1.8 μl of 1 m dithiothreitol (final: 30 mm), and the vesicles were solubilized by adding 15 μl of 10% dodecyl β-d-maltoside. CadB was then precipitated with 50 μl of anti-CadB serum and 20 μg of PANSORBIN® cells (Calbiochem). Cells were washed three times with 50 mm sodium phosphate, pH 7.5, containing 0.1 m NaCl and 0.02% dodecyl β-d-maltoside. Cells were suspended in 40 μl of SDS sample buffer (25 mm Tris-HCl, pH 6.8, 5% glycerol, 1% sodium dodecyl sulfate, 0.05% bromphenol blue) and boiled for 4 min. After centrifugation, a 30-μl aliquot was subjected to SDS-poly-acrylamide gel electrophoresis on an 8.5% acrylamide gel. Incorporation of [14C]NEM into CadB was visualized by BAS2000 II imaging analyzer (Fuji Film).Analysis of Hydropathy and Sequence Homology of Proteins—Average hydropathy profiles of the proteins were obtained according to the method of Kyte and Doolittle (28Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17009) Google Scholar) with a hydrophilicity/hydrophobicity plot (GENETYX-MAC Version 10, GENETYX Co.). A model of the secondary structure of proteins was then constructed according to these profiles. Sequence homology of the protein was analyzed by the CLUSTALW V1.83 program (29Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55217) Google Scholar).RESULTSIdentification of Amino Acid Residues Involved in Uptake and Excretion of Cadaverine—It is known that polyamines are recognized by proteins through interaction with acidic and aromatic amino acid residues (7Vassylyev D.G. Tomitori H. Kashiwagi K. Morikawa K. Igarashi K. J. Biol. Chem. 1998; 273: 17604-17609Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 8Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 30Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 31Kashiwagi K. Pistocchi R. Shibuya S. Sugiyama S. Morikawa K. Igarashi K. J. Biol. Chem. 1996; 271: 12205-12208Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). There are 9 aspartic acid and 7 glutamic acid residues in CadB. These residues were individually mutated to asparagine and glutamine, respectively. Cadaverine uptake activity was measured using E. coli JM109 transformed with pMW encoding wild type or mutated CadB. Cadaverine excretion (i.e. cadaverine-lysine antiporter activity) was measured by [14C]cadaverine uptake using lysine-loaded inside-out membrane vesicles prepared from E. coli JM109 transformed with pMW encoding wild type or mutated CadB. As shown in Fig. 1, both uptake and excretion were greatly decreased with the mutants E204Q and D303N and moderately with E76Q and E377Q, and only uptake was moderately decreased with D185N and E408Q. There are 11 tryptophan and 13 tyrosine residues in CadB. These residues were mutated to leucine and the activities were measured. Both uptake and excretion were strongly decreased with the mutants Y73L, Y89L, Y90L, Y235L, and Y423L and moderately with Y55L, Y246L and Y310L, and only uptake was greatly decreased with W43L, Y57L, Y107L, Y366L, and Y368L and moderately with W41L and Y174L (Fig. 1).In our previous studies of PotE, we found that cysteine residues were involved in the recognition of putrescine by PotE (8Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). There are 8 cysteine residues in CadB. Each cysteine was mutated to serine, and uptake and excretion were measured. As shown in Fig. 2, both uptake and excretion were decreased significantly with the mutant C370S, and then with C397S, but mutations C12S, C125S, C196S, C282S, C389S, and C394S had no effect or only modest effects. To confirm that Cys370 is important for CadB activities, all 8 cysteine residues were replaced by serine and both uptake and excretion were measured. As shown in Fig. 3, the activities of the ΔC mutant were decreased greatly, and they were restored only with the mutant ΔC C370, which has one cysteine residue at position 370 but not with the mutants ΔC C12, C125, C196, C282, C389, C394, and C397. The results confirmed that the SH group of Cys370 is involved in both uptake and excretion.FIGURE 2Effect of Cys mutations on the cadaverine uptake and excretion activities of CadB. Assays were performed as described in the legend of Fig. 1 Values are the means ± S.D. of three samples. The amino acid residue (Cys370) indicated by the white letter is involved in both activities.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Necessity of Cys370 residue for the uptake and excretion activities of CadB. Assays were performed as described in the legend of Fig. 1. Values are the means ± S.D. of three samples. The amino acid residue (Cys370) indicated by the white letter is involved in both activities.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The largest effects on both uptake and excretion of cadaverine were seen with the CadB mutants E204Q, D303N, Y73L, Y89L, Y90L, Y235L, and Y423L (Fig. 1). To confirm the importance of these amino acid residues, Glu204 was replaced by Asp and Asp303 by Glu. As shown in Fig. 4A, replacement of Glu204 and Asp303 by Asp and Glu did not restore the activities of CadB, indicating that Glu204 and Asp303 are critical for both activities. Similarly, Tyr73, Tyr89, Tyr90, Tyr235, and Tyr423 were replaced by Phe and Trp. Replacement of Tyr73, Tyr89, and Tyr235 by Phe and Trp did not restore the activities of CadB. Replacement of Tyr90 by Phe, but not by Trp, and replacement of Tyr423 by Phe and Trp slightly restored the activities of CadB. These results also indicate that these five tyrosine residues are critical for both activities of CadB. Expression of mutated CadB proteins was examined by Western blot analysis using 30 μg of protein of inside-out membrane vesicles. Comparable amounts of mutated CadB proteins were expressed on the membranes, indicating that the mutations do not affect expression of CadB (Fig. 4B).FIGURE 4Cadaverine uptake and excretion activities of Glu204, Asp303, Tyr73, Tyr89, Tyr90, Tyr235 and Tyr423 mutants of CadB (A) and their expression (B). A, assays were performed as described in the legend of Fig. 1. Amino acid residues shown in the horizontal axis are the replaced amino acid residues. Values are the mean ± S.D. of three samples. B, Western blot analysis was performed using 30 μg of protein of the inside-out membrane vesicles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Km values for uptake and excretion of cadaverine by mutants were then determined by Lineweaver-Burk plots. As shown in Table 1, the Km value for uptake of cadaverine by wild-type CadB was 20.8 μm. In mutants that caused a reduction in activity, the Km value for cadaverine was increased, and the change in Km paralleled the decrease in cadaverine uptake activity shown in Figs. 1 and 2. As a control, the Km values were measured with W269L and D436N mutants, whose activities were almost the same as that of wild type. They were nearly equal to the Km value of wild type. These results indicate that all of the key mutants are at amino acid residues involved in the recognition of cadaverine. The Km value for excretion of cadaverine by CadB was 390 μm (Table 1). In CadB mutants that caused a decrease in excretion, the Km value for cadaverine was increased, in parallel with the decrease in cadaverine excretion shown in Figs. 1 and 2. Although the change in the Km value for uptake of cadaverine was greater than the change in the Km value for excretion of cadaverine for any given mutant except the mutants Y73L and Y423, the change in the Km value for uptake was parallel with that for excretion.TABLE 1The Km value of mutated CadB for cadaverine uptake and excretionMutantUptakeaAssays were performed as described in the legend of Fig. 1.ExcretionaAssays were performed as described in the legend of Fig. 1.KmbThese values were calculated from the Lineweaver-Burk plots. Values are mean ± S.D. of three samples.Degree of increaseKmbThese values were calculated from the Lineweaver-Burk plots. Values are mean ± S.D. of three samples.Degree of increaseμM-foldmM-foldWild20.8 ± 0.460.39 ± 0.01Y55L68.0 ± 8.233.271.02 ± 0.062.62Y73L193 ± 19.29.283.86 ± 0.069.90E76Q89.4 ± 3.064.301.65 ± 0.144.23Y89L223 ± 27.910.72.05 ± 0.135.26Y90L155 ± 13.57.452.37 ± 0.276.08E204Q456 ± 65.321.92.67 ± 0.386.85Y235L490 ± 10923.62.82 ± 0.267.23Y246L75.2 ± 6.933.621.30 ± 0.053.33D303N511 ± 5.1524.62.96 ± 0.428.97Y310L81.1 ± 10.43.901.10 ± 0.102.82C370A55.5 ± 0.972.670.99 ± 0.182.54E377Q78.3 ± 3.613.760.72 ± 0.081.85Y423L111 ± 9.265.342.61 ± 0.946.69W269L21.2 ± 0.421.020.41 ± 0.051.05D436N21.0 ± 0.321.010.45 ± 0.081.15W41L35.2 ± 1.021.69W43L111 ± 2.165.34Y57L127 ± 9.456.11Y107L143 ± 11.26.88Y174L50.0 ± 4.292.40D185N86.2 ± 6.104.14Y366L323 ± 35.315.5Y368L111 ± 9.265.34E408Q67.1 ± 4.453.23a Assays were performed as described in the legend of Fig. 1.b These values were calculated from the Lineweaver-Burk plots. Values are mean ± S.D. of three samples. Open table in a new tab For the related protein PotE, a putrescine-ornithine anti-porter, residue Lys301 in C5 (cytoplasmic loop 5) is involved in recognition of the carboxyl group of ornithine (8Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Thus, we determined whether a basic amino acid(s) in the cytoplasmic loops is involved in recognition of the carboxyl group of lysine. As shown in Fig. 5, excretion but not uptake of cadaverine was markedly reduced in R299A in C5, whereas both uptake and excretion activities were reduced to a similar, small extent in all other mutants. The results suggest that Arg299 is involved in the recognition of lysine.FIGURE 5Effect of Arg and Lys mutations on the cadaverine uptake and excretion activities of CadB. Arg and Lys located in the cytoplasmic loops were mutated to Ala. Assays were performed as described in the legend of Fig. 1. Values are the mean ± S.D. of three samples. The amino acid residues in rectangles are involved in lysine recognition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Location of Amino Acid Residues Involved in Uptake and Excretion—Average hydropathy profiles of CadB indicated that CadB consists of 12 helices like PotE. Sequence homology between CadA and PotE was 30.7% (12Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (149) Google Scholar). To confirm the high sequence similarity between CadB and PotE, the multiple sequence alignments were analyzed by the CLUSTALW program (29Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55217) Google Scholar). The score between PotE and CadB was 27.3, but the scores between PotE and other proteins were less than 16. Thus, the secondary structure of CadB was constructed as shown in Fig. 6, which is similar to that of PotE. Amino acids important for both uptake and excretion were located in the transmembrane helices II, III, VI, VII, X, and XII or in the cytoplasmic region between transmembrane helices II and III (C2), VI and VII (C4), VIII and IX (C5), and X and XI (C6). Amino acid residues in the transmembrane helices are also located in the cytoplasmic side rather than the periplasmic side. In contrast to PotE, the Km value for cadaverine uptake by CadB is high (20.8 μm). Since numerous amino acid residues are involved in uptake of cadaverine, the participation of any individual residue in recognition of cadaverine may be weak. Those amino acids are mainly located in the transmembrane helices and the periplasmic loops and may be slightly separated from the substrate binding site. However, it is expected that those amino acid are located close to each other in the tertiary structure of CadB.FIGURE 6Amino acid residues involved in the activities of CadB. A model of the secondary structure of proteins was constructed according to the average hydropathy profiles obtained with a Hydrophilicity/Hydrophobicity Plot (GENETYX-MAC Version 10). Putative transmembrane segments are shown in large boxes. Amino acid residues involved in cadaverine uptake activity and both uptake and excretion activities are classified by symbols shown in the figure. The large and small circles indicate strong and moderate involvement in the activities, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transmembrane Helix Packing of CadB—It has been reported that a hydrophilic cavity of lactose permease having twelve transmembrane helices is formed between eight transmembrane helices: I, II, IV, V, VII, VIII, X, and XI (32Abr" @default.
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• W2065291275 title "Identification of the Cadaverine Recognition Site on the Cadaverine-Lysine Antiporter CadB" @default.
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