SummaryRND (Resistance-Nodulation-Division) family transporters are widespread especially among Gramnegative bacteria, and catalyze the active efflux of many antibiotics and chemotherapeutic agents. They have very large periplasmic domains, and form tripartite complexes with outer membrane channels and periplasmic adaptor proteins. AcrAB-TolC complex of Escherichia coli, which pumps out a very wide range of drugs, has been studied most intensively. Early studies showed that the transporter captures even those substrates that cannot permeate across the cytoplasmic membrane, such as dianionic β-lactams, suggesting that the capture can occur from the periplasm. It was also suggested that the capture occurs from the cytoplasmic membrane/periplasm interface, because most substrates contain a sizable hydrophobic domain; however, this may simply be a reflection of the nature of the binding site within AcrB. Genetic studies of chimeric transporters showed that much of the substrate specificity is determined by their periplasmic domains. Biochemical studies with intact cells recently led to the determination of the kinetic constants of AcrB for some β-lactams, and the result confirms the old prediction that AcrB is a rather slow pump. Reconstitution of purified AcrB and its relatives showed that the pump is a drug/proton antiporter, that AcrA strongly stimulates the activity of the pump, and that AcrB seems to have a highest affinity for conjugated bile salts. Structural study with mutants of the network of charged residues in the transmembrane domain showed that protonation here produced a far-reaching conformational change, which was found to be present in one of the protomers in the asymmetric crystal structure of the wild-type AcrB. The functional rotatory hypothesis then predicts that the drug bound in the periplasmic domain is extruded through this conformational change initiated by the protonation of one of the residues in the aforementioned network, an idea that was recently supported by disulfide cross-linking as well as by the behavior of linked AcrB protomers.

The AcrB trimeric multidrug efflux transporter of Escherichia coli pumps out a very wide spectrum of compounds. Although minocycline and doxorubicin have been cocrystallized within the large binding pocket in the periplasmic domain of the binding protomer, nothing is known about the binding of many other ligands to this protein. We used computer docking to evaluate the interaction of about 30 compounds with the binding protomer and found that many of them are predicted to bind to a narrow groove at one end of the pocket whereas some others prefer to bind to a wide cave at the other end. Competition assays using nitrocefin efflux and covalent labeling of Phe615Cys mutant AcrB with fluorescein-5-maleimide showed that presumed groove-binders competed against each other, but cave-binders did not compete against groove-binders, although the number of compounds tested was limited. These results give us at least a hypothesis to be tested by more biochemical and genetic experiments in the future.

We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.

Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The "linked-trimer" AcrB was expressed well in the inner membrane fraction of ⌬acrB ⌬recA strains, as a large protein of ϳ300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.RND (resistance-nodulation-cell division) family (23) multidrug efflux transporters, such as AcrB of Escherichia coli, not only are responsible for the intrinsic resistance of gram-negative bacteria to many lipophilic agents but also, when overproduced, generate a multidrug-resistant phenotype (13) that is becoming a major clinical problem in organisms like Pseudomonas aeruginosa (25). AcrB has been studied most extensively as a prototype among these RND multidrug transporters and is especially interesting as it allows the extrusion of an extremely wide range of substrates, including basic dyes; antibiotics such as chloramphenicol, tetracyclines, novobiocin, macrolides, and ␤-lactams; detergents such as sodium dodecyl sulfate (SDS) and Triton X-100; and even simple solvents (8, 13). AcrB exists as a homotrimer in which each subunit contains 12 transmembrane helices (TM1 to TM12) and two large periplasmic domains between TM1 and TM2 and between TM7 and TM8 (12). Recent elucidation of the asymmetric trimer structure through X-ray crystallography (11,17,19) led to the notion that the transporter works by a functionally rotating mechanism in which each protomer goes through a cycle of conformational alterations which are facilitated in turn by the complementary alterations in neighboring protomers.Although t...

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals and expels them from cells. It is a proton antiporter belonging to the resistance-nodulation-division (RND) superfamily. Asp407, Asp408, Lys940, and Arg971 in transmembrane (TM) helices of this transporter have been identified as essential amino acid residues that probably function as components of the proton relay system. In this study, we identified a novel residue in TM helix 11, Thr978, as an essential residue by alanine scanning mutagenesis. Its location close to Asp407 suggests that it is also a component of the proton translocation pathway, a prediction confirmed by the similar conformations adopted by T978A, D407A, D408A, and K940A mutant proteins (see the accompanying paper). Sequence alignment of 566 RND transporters showed that this threonine residue is conserved in about 96% of cases. Our results suggest the hypotheses that Thr978 functions through hydrogen bonding with Asp407 and that protonation of the latter alters the salt bridging and hydrogen bonding pattern in the proton relay network, thus initiating a series of conformational changes that ultimately result in drug extrusion.Multidrug transporters cause serious problems in the chemotherapy of cancer as well as in the antibiotic treatment of bacterial infections. These membrane proteins recognize many structurally dissimilar toxic compounds and actively extrude them from the cell. The Escherichia coli AcrB transporter (10, 11), which is a member of the resistance-nodulation-division (RND) family of transporters (26), is responsible for most of the intrinsic drug resistance of this organism (14,15,22) and is one of the best-studied multidrug pumps. It occurs as a multiprotein complex (24,25,32), with an outer membrane channel TolC protein (4, 9) and a periplasmic linker protein, AcrA (10), and this complex structure allows the direct export of drugs to the external medium (14). The structural work of Murakami et al. (12) revealed that unbound AcrB is a homotrimer, where each subunit contains 12 transmembrane (TM) helices and two large periplasmic domains, between TM1 and TM2 and between TM7 and TM8. The top of the periplasmic domain of AcrB is thought to associate with TolC.AcrB utilizes proton motive force (PMF) as energy for its transport function (10,30,31). The molecular mechanism that couples proton translocation to the efflux of drugs has not been elucidated completely. However, the simplest mechanism seems to operate in a small multidrug transporter, EmrE, of E. coli (19,23,28). In this transporter, there exists only one membrane-embedded charged residue, Glu14, and this residue is involved in the recognition of both the substrates and the coupling proton. For another well-studied PMF-dependent transporter, the lactose permease LacY from E. coli (1), the proton translocation pathway involves several charged residues in the TM helices and is separate from the substrate transport pathway.Charged residues embedded in the membrane were also show...

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.It is now well known that multidrug efflux transporters cause problems in cancer chemotherapy, as well as in antibiotic treatment of bacterial infections. These transporters recognize many structurally dissimilar toxic compounds and actively extrude them from the cell. The Escherichia coli AcrB transporter (14, 15), which belongs to the resistance-nodulationdivision family (32), is responsible for most of the intrinsic drug resistance of this organism (20,21,28) and is also perhaps the best-studied bacterial multidrug pump. It is a homotrimer in which each subunit contains 12 transmembrane (TM) helices and two large periplasmic domains, one between TM helix 1 (TM1) and TM2 and one between TM7 and TM8. The trimeric AcrB molecule in turn occurs as a multiprotein complex (30,31,36) together with the outer membrane channel protein TolC (8, 12) and the periplasmic linker protein AcrA (14). The top of the periplasmic domain of AcrB is thought to interact with the internal end of TolC, and this structure allows direct export of drugs to the external medium (20).AcrB utilizes the proton motive force as energy for its transport function (14,34,35). Charged residues within the TM helices of this transporter, including Asp407, Asp408, and Lys940, have been identified as amino acid residues essential for activity (19), presumably functioning as components of the proton relay system. We previously...

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated ldc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.There are two types of bacterial amino acid decarboxylase, constitutive and inducible. The former type includes decarboxylases for L-ornithine, L-arginine, S-adenosyl-L-methionine, and diaminopimelic acid (27). In Escherichia coli, a previously characterized lysine decarboxylase (EC 4.1.1.18) is encoded by cadA at 93.7 min and participates in the synthesis of cadaverine from lysine. This enzyme is inducible under anaerobic conditions at pH 5.5 and by adding lysine to the culture medium (7,19,23,27). Some evidence has been presented for the existence of a second, much less active, constitutive lysine decarboxylase in E. coli (6, 30), but the data are still too incomplete to permit any definitive conclusions on the presence of this second enzyme. Igarashi et al. made several observations concurring with the suggestion that cadaverine is actually formed by ornithine decarboxylase, which would then account for the alleged constitutive lysine decarboxylase (8). The presence of constitutive lysine decarboxylase has not been detected in organisms, except for Selenomonas ruminantium, a strictly anaerobic gram-negative bacterium (12).We previously reported the overproduction of lysine by strain WC196, a lysine analog (S-aminoethyl-L-cysteine)-resistant strain of W3110 formed by N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenesis. During the construction of strains derived from this strain, we found that a cadA deletion mutant (WC196C) could still degrade lysine to cadaverine (15a). This finding suggested the existence of another lysine decarboxylase besides cadA in E. coli.Here, we report the existence of a new lysine decarboxylase gene, designated ldc, at 4.7 min on the E. coli chromosome, and we describe the lysine decarboxylase activities of strains containing cadA, ldc, or cadA ldc mutations. MATERIALS AND METHODSBacterial strains, bacteriophag...

). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17), including the mouse, Saccharomyces cerevisiae, Neurospora crassa, Trypanosoma brucei, and Caenorhabditis elegans enzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved in S. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward both L-lysine and L-ornithine with a substrate specificity ratio of 0.83 (defined as the k cat /K m ratio obtained with L-ornithine relative to that obtained with L-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/ P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an important enzyme for the biosynthesis of putrescine, a precursor of polyamines which are implicated in a wide variety of biological processes that include the synthesis of DNA, RNA, and protein in all living cells (26,30,31). Lysine decarboxylase (LDC) (EC 4.1.1.18), which exists in most bacteria, is involved in the biosynthesis of cadaverine, a molecule that participates in the closing of the porin channels in the outer membrane of Escherichia coli (5) and is also an essential component of the peptidoglycan of Selenomonas ruminantium, Veillonella alcalescens, V. parvula, and Anaerovibrio lipolytica, which are strictly anaerobic gram-negative bacteria (9,12,14,15). Previously, we reported that in these bacteria, cadaverine is transferred to the D-glutamic acid residue of a lipid intermediate for the synthesis of the cadaverine-containing peptidoglycan by cadaverine transferase (11,12,15,17). In S. ruminantium, cadaverine is constitutively synthesized from L-lysine (16) and its synthesis was completely prevented by DL-␣-difluoromethyllysine (DFML) and DL-␣-difluoromethylornithine (DFMO), which are irreversible inhibitors of LDC from Mycoplasma dispar (27) and eukaryotic ODC, respectively, resulting in growth inhibition due to the synthesis of the abortive peptidoglycan without cadaverine (11,15). These observations suggested that S. ruminantium ODC could decarboxylate L-lysine, as well as Lornithine. Accordingly,...