Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Yersinia pestis, the causative agent of plague, two inorganic iron transport systems have been partially characterized. The yersiniabactin (Ybt) system is a siderophore-dependent transport system required for full virulence. Yfe is an ABC transport system that accumulates both iron and manganese. We have identified and cloned a Y. pestis yfuABC operon. The YfuABC system is a member of the cluster of bacterial ABC iron transporters that include Sfu of Serratia, Hit of Haemophilus, and Yfu of Yersinia enterocolitica. The Y. pestis KIM6+ system is most homologous to that in Y. enterocolitica, showing identities of 84% for YfuA (periplasmic binding protein), 87% for YfuB (inner membrane permease), and 75% for YfuC (ATP hydrolase). We constructed a yfuABC promoter-lacZ fusion to examine regulation of transcription. This promoter contains a potential Fur binding sequence and is iron and Fur regulated. Significant expression from the yfuABC promoter occurred during iron-deficient growth conditions. In vitro transcription and translation of a recombinant plasmid encoding yfuABC indicates that YfuABC proteins are expressed. Escherichia coli 1017 (an enterobactin-deficient mutant) carrying this plasmid was able to grow in an iron-restrictive complex medium. We constructed a deletion encompassing the yfuABC promoter and most of yfuA. This mutation was introduced into strains with mutations in Ybt, Yfe, or both systems to examine the role of Yfu in iron acquisition in Y. pestis. Growth of the yfu mutants in a deferrated, defined medium (PMH2) at 26 and 37 degrees C failed to identify a growth or iron transport defect due to the yfu mutation. Fifty percent lethal dose studies in mice did not demonstrate a role for the Yfu system in mammalian virulence.
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PMID:Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system. 1129 95

The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.
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PMID:The pleitropic drug ABC transporters from Saccharomyces cerevisiae. 1132 75

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.
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PMID:Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers. 1134 49

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.
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PMID:Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. 1137 44

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.
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PMID:The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter. 1140 22

The polyamine content of cells is regulated by biosynthesis, degradation, and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) are ABC transporters, each consisting of a periplasmic substrate binding protein, two transmembrane proteins, and a membrane-associated ATPase. The third transport system, catalyzed by PotE, mediates both uptake and excretion of putrescine. In this article, the properties of the first two polyamine uptake systems are reviewed in detail.
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PMID:Polyamine uptake systems in Escherichia coli. 1142 Dec 74

In both bacteria and archaea, molybdate is transported by an ABC-type transporter comprising three proteins, ModA (periplasmic binding protein), ModB (membrane protein) and ModC, the ATPase. The modABC operon expression is controlled by ModE-Mo. In the absence of the high-affinity molybdate transporter, molybdate is also transported by another ABC transporter which transports sulfate/thiosulfate as well as by a nonspecific anion transporter. Comparative analysis of the molybdate transport proteins in various bacteria and archaea is the focus of this review.
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PMID:Molybdate transport. 1142 Dec 78

ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A. Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity. The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer. We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents.
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PMID:Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. 1143 80

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.
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PMID:Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing. 1153 60

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.
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PMID:N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli. 1170 Mar 59


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