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Yoshimi Kakinuma - One of the best experts on this subject based on the ideXlab platform.
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rotation mechanism of Enterococcus hirae v 1 atpase based on asymmetric crystal structures
Nature, 2013Co-Authors: Yoshimi Kakinuma, Satoshi Arai, Shinya Saijo, Kano Suzuki, Kenji Mizutani, Yoshiko Ishizukakatsura, Noboru OhsawaAbstract:Several crystal structures of the rotary motor of bacterial V-ATPase are solved at high resolution, representing different asymmetric structures and enabling the prediction of a model for the rotational mechanism of V1-ATPase. Vacuolar-type H+-ATPases (V-ATPases) are biomolecular rotary motors that couple ATP hydrolysis to the transport of a proton across intracellular and plasma membranes of eukaryotic cells. V-ATPase functions in many cellular processes and is an important drug target for diseases such as osteoporosis and cancer. This paper reports several X-ray crystal structures of a V1-ATPase from Enterococcus hirae, which reveal the conformational changes that occur when ATP binds the protein. On the basis of these structures the authors propose a model for the rotation mechanism of this membrane protein. In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer1. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V1-ATPase from the A3B3 and DF complexes2,3. Here we report the asymmetric structures of the nucleotide-free (2.8 A) and nucleotide-bound (3.4 A) A3B3 complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 A) and nucleotide-bound (2.7 A) V1-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V1-ATPase.
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significance of the glutamate 139 residue of the v type na atpase ntpk subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae
Journal of Bacteriology, 2011Co-Authors: Miyuki Kawanokawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Significance of the glutamate-139 residue of the V-type Na+-ATPase NtpK subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae.
Journal of bacteriology, 2011Co-Authors: Miyuki Kawano-kawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Expression and purification of the central stalk subunits of Na + -translocating V-type ATPase from Enterococcus hirae
African Journal of Biotechnology, 2011Co-Authors: Kmm Hossain, Yoshimi Kakinuma, Takeshi Murata, Satoshi Arai, Shinya Saijo, Ichiro YamatoAbstract:Enterococcus hirae (E. hirae) vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V 1 ; NtpA 3 -B 3 -D-G) and an integral membrane domain (V o ; NtpI-K 10 ) connected by a central and peripheral stalks. Central stalk of Na + -translocating V-type ATPase of E. hirae is composed of NtpC, NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na + -ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, into the plasmid vector, pET23d. NtpC, NtpD and NtpG subunit proteins were expressed, separately as His-tagged soluble proteins in Escherichia coli BL21(DE3) cells and then, purified by Ni Sepharose 6 fast flow column. Purification of expressed protein was confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The amount of purified NtpC, NtpD and NtpG subunit proteins were measured as 14, 17 and 15 mg/1 liter culture, respectively. Key words: Enterococcus hirae, V-ATPase, central stalk subunits, expression.
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Deletion analysis of the subunit genes of V-type Na+-ATPase from Enterococcus hirae
Journal of biochemistry, 2006Co-Authors: Toshiaki Hosaka, Yoshimi Kakinuma, Takeshi Murata, Kazuma Takase, Ichiro YamatoAbstract:The V 1 V o -ATP ase from Enterococcus hirae catalyzes ATP hydrolysis coupled with sodium translocation. Mutants with deletions of each of 10 subunits (NtpA, B, C, D, E, F, G, H, I, and K) were constructed by insertion of a chloramphenicol acetyltransferase gene into the corresponding subunit gene in the genome. Measurements of cell growth rates, 22 Na + efflux activities, and ATP hydrolysis activities of the membranes of the deletion mutants indicated that V-ATPase requires nine of the subunits, the exception being the NtpH subunit. The results of Western blotting and V 1 -ATPase dissociation analysis suggested that the A, B, C, D, E, F, and G subunits constitute the V 1 moiety, whereas the V 0 moiety comprises the I and K subunits.
Takeshi Murata - One of the best experts on this subject based on the ideXlab platform.
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Rotational mechanism of Enterococcus hirae V1-ATPase by crystal-structure and single-molecule analyses.
Current opinion in structural biology, 2015Co-Authors: Ryota Iino, Yoshihiro Minagawa, Hiroshi Ueno, Kano Suzuki, Takeshi MurataAbstract:In ion-transporting rotary ATPases, the mechanical rotation of inner rotor subunits against other stator subunits in the complex mediates conversion of chemical free energy from ATP hydrolysis into electrochemical potential by pumping ions across the cell membrane. To fully understand the rotational mechanism of energy conversion, it is essential to analyze a target sample by multiple advanced methods that differ in spatiotemporal resolutions and sample environments. Here, we describe such a strategy applied to the water-soluble V1 moiety of Enterococcus hirae V-ATPase; this strategy involves integration of crystal structure studies and single-molecule analysis of rotary dynamics and torque generation. In addition, we describe our current model of the chemo-mechanical coupling scheme obtained by this approach, as well as future prospects.
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Torque generation of Enterococcus hirae V-ATPase.
The Journal of biological chemistry, 2014Co-Authors: Hiroshi Ueno, Takeshi Murata, Ichiro Yamato, Yoshihiro Minagawa, Mayu Hara, Suhaila Rahman, Eiro Muneyuki, Hiroyuki Noji, Ryota IinoAbstract:V-ATPase (VoV1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in VoV1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae VoV1 (EhVoV1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhVoV1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhVoV1 only showed the “clear” state without apparent backward steps, whereas EhV1 showed two states, “clear” and “unclear.” Furthermore, EhVoV1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhVoV1 showed faster rotation than EhV1, and the torque of EhVoV1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhVoV1. These results indicate that rotor-stator interactions of the Vo moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhVoV1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhVoV1.
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Molecular structure and rotary dynamics of Enterococcus hirae V1‐ATPase
IUBMB life, 2014Co-Authors: Ryota Iino, Yoshihiro Minagawa, Hiroshi Ueno, Mayu Hara, Takeshi MurataAbstract:V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the high-resolution molecular structure of Enterococcus hirae V1-ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme.
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molecular structure and rotary dynamics of Enterococcus hirae v1 atpase
Iubmb Life, 2014Co-Authors: Takeshi Murata, Ryota Iino, Yoshihiro Minagawa, Hiroshi Ueno, Mayu HaraAbstract:V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the high-resolution molecular structure of Enterococcus hirae V1-ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme.
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significance of the glutamate 139 residue of the v type na atpase ntpk subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae
Journal of Bacteriology, 2011Co-Authors: Miyuki Kawanokawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
Ichiro Yamato - One of the best experts on this subject based on the ideXlab platform.
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Torque generation of Enterococcus hirae V-ATPase.
The Journal of biological chemistry, 2014Co-Authors: Hiroshi Ueno, Takeshi Murata, Ichiro Yamato, Yoshihiro Minagawa, Mayu Hara, Suhaila Rahman, Eiro Muneyuki, Hiroyuki Noji, Ryota IinoAbstract:V-ATPase (VoV1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in VoV1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae VoV1 (EhVoV1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhVoV1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhVoV1 only showed the “clear” state without apparent backward steps, whereas EhV1 showed two states, “clear” and “unclear.” Furthermore, EhVoV1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhVoV1 showed faster rotation than EhV1, and the torque of EhVoV1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhVoV1. These results indicate that rotor-stator interactions of the Vo moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhVoV1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhVoV1.
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significance of the glutamate 139 residue of the v type na atpase ntpk subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae
Journal of Bacteriology, 2011Co-Authors: Miyuki Kawanokawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Significance of the glutamate-139 residue of the V-type Na+-ATPase NtpK subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae.
Journal of bacteriology, 2011Co-Authors: Miyuki Kawano-kawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Expression and purification of the central stalk subunits of Na + -translocating V-type ATPase from Enterococcus hirae
African Journal of Biotechnology, 2011Co-Authors: Kmm Hossain, Yoshimi Kakinuma, Takeshi Murata, Satoshi Arai, Shinya Saijo, Ichiro YamatoAbstract:Enterococcus hirae (E. hirae) vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V 1 ; NtpA 3 -B 3 -D-G) and an integral membrane domain (V o ; NtpI-K 10 ) connected by a central and peripheral stalks. Central stalk of Na + -translocating V-type ATPase of E. hirae is composed of NtpC, NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na + -ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, into the plasmid vector, pET23d. NtpC, NtpD and NtpG subunit proteins were expressed, separately as His-tagged soluble proteins in Escherichia coli BL21(DE3) cells and then, purified by Ni Sepharose 6 fast flow column. Purification of expressed protein was confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The amount of purified NtpC, NtpD and NtpG subunit proteins were measured as 14, 17 and 15 mg/1 liter culture, respectively. Key words: Enterococcus hirae, V-ATPase, central stalk subunits, expression.
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Deletion analysis of the subunit genes of V-type Na+-ATPase from Enterococcus hirae
Journal of biochemistry, 2006Co-Authors: Toshiaki Hosaka, Yoshimi Kakinuma, Takeshi Murata, Kazuma Takase, Ichiro YamatoAbstract:The V 1 V o -ATP ase from Enterococcus hirae catalyzes ATP hydrolysis coupled with sodium translocation. Mutants with deletions of each of 10 subunits (NtpA, B, C, D, E, F, G, H, I, and K) were constructed by insertion of a chloramphenicol acetyltransferase gene into the corresponding subunit gene in the genome. Measurements of cell growth rates, 22 Na + efflux activities, and ATP hydrolysis activities of the membranes of the deletion mutants indicated that V-ATPase requires nine of the subunits, the exception being the NtpH subunit. The results of Western blotting and V 1 -ATPase dissociation analysis suggested that the A, B, C, D, E, F, and G subunits constitute the V 1 moiety, whereas the V 0 moiety comprises the I and K subunits.
Kazuei Igarashi - One of the best experts on this subject based on the ideXlab platform.
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significance of the glutamate 139 residue of the v type na atpase ntpk subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae
Journal of Bacteriology, 2011Co-Authors: Miyuki Kawanokawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Significance of the glutamate-139 residue of the V-type Na+-ATPase NtpK subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae.
Journal of bacteriology, 2011Co-Authors: Miyuki Kawano-kawada, Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Hiroko Takahashi, Michio Homma, Yoshimi KakinumaAbstract:A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
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Isolation of Enterococcus hirae Mutant Deficient in Low-affinity Potassium Uptake at Alkaline pH
Bioscience biotechnology and biochemistry, 2002Co-Authors: Miyuki Kawano, Kazuei Igarashi, Yoshimi KakinumaAbstract:We here isolated an Enterococcus hirae mutant unable to grow well at pH 10. The influx rate calculated from steady-state 42K+/K+ exchange and the intracellular K+ concentration of the mutant were reduced to 53 and 55% of those of the wild-type, respectively. The activities of two high-affinity K+ uptake systems, KtrI and KtrII, were normal in the mutant, but the kinetics of net K+ uptake at pH 10 indicated that a low-affinity K+ uptake with a Km of about 20 mM (Kawano, M, Abuki, R, Igarashi, K, Kakinuma, Y. (2001) Arch. Microbiol. 175: 41-45), which were seen in the wild-type, was deficient in this mutant.
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Catalytic properties of Na(+)-translocating V-ATPase in Enterococcus hirae.
Biochimica et biophysica acta, 2001Co-Authors: Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Miyuki Kawano, Yoshimi KakinumaAbstract:V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na a -translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na a -ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K a transporter independent of the Na a -ATPase. Expression of this operon, encoding two transport systems for Na a and K a ions, was regulated at the transcriptional level by intracellular Na a as the signal. Structural aspects and catalytic properties of purified Na a -ATPase closely resembled those of other V-type H a -ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na a at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na a binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na a binding sites of the enzyme. fl 2001 Elsevier Science B.V. All rights reserved.
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catalytic properties of na translocating v atpase in Enterococcus hirae
Biochimica et Biophysica Acta, 2001Co-Authors: Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Miyuki Kawano, Yoshimi KakinumaAbstract:V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na a -translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na a -ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K a transporter independent of the Na a -ATPase. Expression of this operon, encoding two transport systems for Na a and K a ions, was regulated at the transcriptional level by intracellular Na a as the signal. Structural aspects and catalytic properties of purified Na a -ATPase closely resembled those of other V-type H a -ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na a at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na a binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na a binding sites of the enzyme. fl 2001 Elsevier Science B.V. All rights reserved.
Marc Solioz - One of the best experts on this subject based on the ideXlab platform.
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Genome Sequence of Enterococcus hirae (Streptococcus faecalis) ATCC 9790, a Model Organism for the Study of Ion Transport, Bioenergetics, and Copper Homeostasis
Journal of bacteriology, 2012Co-Authors: Thomas Gaechter, Christof Wunderlin, Tobias Schmidheini, Marc SoliozAbstract:Enterococcus hirae ATCC 9790 is a Gram-positive lactic acid bacterium that has been used in basic research for over 4 decades. Here we report the sequence and annotation of the 2.8-Mb genome of E. hirae and its endemic 29-kb plasmid pTG9790.
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Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae.
Biometals : an international journal on the role of metal ions in biology biochemistry and medicine, 2005Co-Authors: David Magnani, Marc SoliozAbstract:Extensive insight into copper homeostasis has recently emerged. The Gram-positive bacterium Enterococcus hirae has been a paradigm for many aspects of the process. The cop operon of E. hirae consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. CopA and CopB accomplish copper uptake and export, respectively, and the expression of the cop operon is regulated by copper via the CopY repressor and the CopZ chaperone. The functions of the four Cop proteins have been extensively studied in vivo as well as in vitro and a detailed understanding of the regulation of the cop operon by copper has emerged.
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Copper homeostasis in Enterococcus hirae.
FEMS microbiology reviews, 2003Co-Authors: Marc Solioz, Jivko V StoyanovAbstract:Copper is an essential component of life because of its convenient redox potential of 200-800 mV when bound to protein. Extensive insight into copper homeostasis has only emerged in the last decade and Enterococcus hirae has served as a paradigm for many aspects of the process. The cop operon of E. hirae regulates copper uptake, availability, and export. It consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. Most of these components have been conserved across the three evolutionary kingdoms. The four Cop proteins have been studied in vivo as well as in vitro and their function is understood in some detail.
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Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase.
FEBS letters, 2001Co-Authors: Karl-dimiter Bissig, Thomas C Voegelin, Marc SoliozAbstract:Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC50 of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.
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Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae.
Biochemical and biophysical research communications, 2001Co-Authors: Haibo Wunderli-ye, Marc SoliozAbstract:The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a Vmax of 0.15 μmol/min/mg and a Km for ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro.