Thiamin transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide

Active transport of thiamin (vitamin B₁) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K_M=15 nM and V_max=46 U mg⁻¹. Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V_max by approximately 50% without effecting K_M or the apparent first-order rate constant (k_obsd). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.     

Correlation between Multi-Drug Resistance-Associated Membrane Transport in Clonal Cancer Cells and the Cell Cycle Phase

Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters, V(max) and K(M). Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater V(max) values, indicating a higher activity of transport. They also exhibited a greater V(max)/K(M) ratio, indicating a higher efficiency of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents.     

Overexpression,purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli

Thiamin (Vitamin B(1)) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity. A subsequent biochemical characterization demonstrates that TBP is a 34.205kDa monomer. TBP also contains one tightly bound thiamin species [thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)] per monomer (K(D)=0.8 microM) when isolated under conditions that would remove any loosely bound ligands. We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k(off)=0.12s(-1). The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E. coli. The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.     

Shape and dynamics of thermoregulating honey bee clusters

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.     

Lipase-catalyzed synthesis of isoamyl butyrate. A kinetic study.

Kinetics of lipase-catalyzed esterification of butyric acid and isoamyl alcohol have been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. No evidence of any significant diffusional limitations was detected that could affect the kinetics. The values of the apparent kinetic parameters were computed as: V(max)=11.72 micromol/min/mg; K(M, Acid)=0.00303 M; K(M, Alcohol)=0.00306 M; K(i, Acid)=1.05 M; and K(i, Alcohol)=6.55 M. This study indicates a competitive enzyme inhibition by butyric acid during lipase-catalyzed esterification reaction. Butyric acid, being a short-chain polar acid, concentrates in the microaqueous layer and causes a pH drop in the enzyme microenvironment leading to enzyme inactivation. Butyric acid binds to acyl-enzyme complex unproductively to yield a dead-end intermediate that can no longer give rise to an ester. High concentration of butyric acid gave rise to inactivation of the biocatalyst in addition to dead-end inhibition.     

Glucose Transport in Brucella abortus

Brucella abortus British strain 19 transported glucose with an apparent K(m) of 0.16 mM and an apparent V(max) of 250 nmol per min per mg of N. The only common glucose analogue transported was 2-deoxyglucose (2-DOG), with an apparent K(i) of 0.73 mM. Alpha- or beta-methyl glucosides and 3-O-methylglucose were not transported. Transport was linear for 70 to 90 s, depending on the concentration of substrate used. 2-Deoxyglucose was transported as the free sugar and was not further metabolized once inside the cell. There was no glucose phosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and there were no inhibitors present in Brucella cell-free extract that inhibited the Escherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (pCMB) completely inhibited transport of glucose and 2-DOG. Glutathione, dithiothreitol, and beta-mercaptoethanol reversed the effects of pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of 37 to 45 C were observed for both K(m) and V(max). The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, erythritol, or glucose. The electron transfer inhibitors carbonyl cyanide, m-chlorophenylhydrazone, NaN(3), 2,4-dinitrophenol, and KCN inhibited 2-DOG transport to a greater extent than did the metabolic energy inhibitors NaAsO(4), iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatases, inhibited transport by 100%.     

The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter

The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.     

Maltose and maltodextrin utilization by Bacillus subtilis

Bacillus subtilis can utilize maltose and maltodextrins that are derived from polysaccharides, like starch or glycogen. In this work, we show that maltose is taken up by a member of the phosphoenolpyruvate-dependent phosphotransferase system and maltodextrins are taken up by a maltodextrin-specific ABC transporter. Uptake of maltose by the phosphoenolpyruvate-dependent phosphotransferase system is mediated by maltose-specific enzyme IICB (MalP; synonym, GlvC), with an apparent K(m) of 5 microM and a V(max) of 91 nmol . min(-1) . (10(10) CFU)(-1). The maltodextrin-specific ABC transporter is composed of the maltodextrin binding protein MdxE (formerly YvdG), with affinities in the low micromolar range for maltodextrins, and the membrane-spanning components MdxF and MdxG (formerly YvdH and YvdI, respectively), as well as the energizing ATPase MsmX. Maltotriose transport occurs with an apparent K(m) of 1.4 microM and a V(max) of 4.7 nmol . min(-1) . (10(10) CFU)(-1).     

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K(m)=379μM and k(cat)=0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Structural similarities between thiamin-binding protein and thiaminase-I suggest a common ancestor

ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.     

Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K(m) value of 1.5 microM and a V(max) of 286 pmol min(-1) (mg protein)(-1) and showed higher affinity for ATP and a lower V(max) value than intact RER (K(m) of 6.5 microM and V(max) of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.     

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.     

Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.     

DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.     

Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA)

The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism. Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K (M)(tRNA)= 1.9 +/- 0.7 microM and K (M)(AdoMet)= 98 +/- 5.0 microM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/- 0.5 microM for sinefungin and S-adenosylhomocysteine, respectively. Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ(1)-tRNA(Tyr) when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ(1)-tRNA(Tyr), but noncompetitive against AdoMet. However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive. Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ(1)-tRNA(Tyr) binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA. The chemical mechanism that we previously proposed for the QueA-catalyzed reaction [Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D. (2000) Org. Lett. 2, 1307-1310] is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme.     

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.     

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 μM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 μM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.     

Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.