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Christopher Brasen - One of the best experts on this subject based on the ideXlab platform.
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identifizierung und charakterisierung der enzyme der acetatbildung und der acetataktivierung in halophilen und hyperthermophilen archaea adp bildende und amp bildende acetyl coa synthetasen aus Haloarcula marismortui und pyrobaculum aerophilum
2006Co-Authors: Christopher BrasenAbstract:Acetat ist eines der wichtigsten Endprodukte energieliefernder Fermentationsprozesse vieler anaerober oder fakultativer Bacteria und Archaea. Daruber hinaus stellt Acetat aber auch ein weit verbreitetes Substrat fur Katabolismus und Anabolismus aerober und anaerober, pro- und eukaryontischer Mikroorganismen dar. Bisherige Untersuchungen deuten darauf hin, dass der Mechanismus der Acetatbildung in Prokaryonten domanenspezifisch ist. In Bacteria wird die Acetatbildung von zwei Enzymen, der Acetat Kinase (AK) und der Phosphotransacetylase (PTA), katalysiert, wahrend in Archaea die Acetatbildung von nur einem Enzym, der ADP-bildende Acetyl-CoA Synthetase (ACD), erfolgt. Die Hauptmechanismen der Acetataktivierung, der weitverbreitete Mechanismus uber eine AMP-bildende Acetyl-CoA Synthetase (ACS) bzw. der von weniger Organismen beschrittene Mechanismus uber den reversiblen AK/PTA-Weg, kommen dagegen beide sowohl in Bacteria als auch in Archaea vor. Untersuchungen zur Acetatbildung in Archaea und dem daran beteiligten Enzym, der ACD, existierten bisher nur fur wenige hyperthermophile, anaerobe Euryarchaeota. Zudem lagen uber die Acetataktivierung in Archaea, auser fur die methanogenen Methanosaeta und Methanosarcina Spezies, keine Informationen vor. Daher sollten in dieser Arbeit das Wachstum der halophilen Archaea auf Glucose und Acetat untersucht und die Enzyme der Acetatbildung und -aktivierung identifiziert, charakterisiert sowie die kodierenden Gene ermittelt werden. Auserdem sollten die Gene, die fur die entsprechenden Enzyme in dem hyperthermophilen Crenarchaeon Pyrobaculum aerophilum kodieren, identifiziert, kloniert und exprimiert sowie die rekombinanten Proteine biochemisch charakterisiert werden. Identifizierung und Charakterisierung der Enzyme der Acetatbildung und -aktivierung in halophilen Archaea Wachstumsexperimente mit den extrem halophilen Archaea Haloarcula marismortui, Halorubrum saccharovorum, Haloferax volcanii und Halococcus saccharolyticus auf Glucose, Acetat und Glucose/Acetat enthaltenden Medien ergaben, dass die Haloarchaea Glucose und Acetat als Kohlenstoff- und Energiequelle nutzen konnen und dass Glucose gegenuber Acetat das bevorzugte Substrat ist. Zudem konnte mit Enzymmessungen in unterschiedlichen Wachstumsphasen gezeigt werden, dass die Aktivitaten von ACD und ACS substratabhangig reguliert werden. In Phasen der Acetatbildung bei Wachstum auf Glucose stieg die Aktivitat der ACD an, wohingegen die der ACS abnahm. Dagegen stieg in Phasen des Acetatverbrauches umgekehrt die ACS Aktivitat an, die der ACD nahm aber ab. Zusammen mit dem Befund, dass weder AK noch PTA nachgewiesen werden konnten, zeigten diese Experimente deutlich, dass die Acetatbildung in Haloarchaea von einer ACD katalysiert wird, wahrend die Acetataktivierung uber eine induzierbare ACS erfolgt. ACD und ACS wurden aus Ha. marismortui gereinigt und charakterisiert. Die kodierenden Gene wurden identifiziert, kloniert und die rekombinanten Proteine aus Einschlusskorpern reaktiviert und gereinigt. Die ACD ist ein 166 kDa Homodimer und katalysierte die reversible ADP- und Pi-abhangige Acetatbildung aus Acetyl-CoA. Das Enzym benotigte Hochsalzbedingungen (3 M KCl) fur die Stabilitat und setzte neben Acetat auch Propionat, Butyrat, Isobutyrat und Isovalerat, nicht aber die aromatischen Sauren Phenylacetat oder Indol-3-acetat um. Die ACS ist ein 71 kDa Monomer und katalysierte die ATP- und CoA abhangige Umsetzung von Acetat zu Acetyl-CoA, wobei AMP und PPi gebildet werden. Die ACS benotigte 1.25 M KCl fur optimale Aktivitat und setzte bevorzugt Acetat um und zusatzlich nur Propionat mit deutlich geringerer Effizienz. Der Km-Wert der ACS fur Acetat war mit 0.23 mM etwa 10fach kleiner als der der ACD (2.6 mM). Diese hohere Acetataffinitat konnte der Grund fur die Induktion der ACS bei Wachstum auf Acetat sein, weil Acetat v.a. in geringen Konzentrationen, wie sie in hypersalinen Habitaten vorkommen, erheblich effizienter aktiviert werden kann. ADP-bildende und AMP-bildende Acetyl-CoA Synthetasen aus dem hyperthermophilen Crenarchaeon Pyrobaculum aerophilum Im Genom von P. aerophilum wurden Gene identifiziert, die fur eine putative ACD und eine ACS kodieren. Die ORFs wurden exprimiert und die rekombinanten Proteine biochemisch charakterisiert. Es wurde gezeigt, dass die ORFs PAE3250 und PAE3249 aus P. aerophilum, die fur eine funktionelle ACD kodierten, eine fur bisher charakterisierte ACD ungewohnliche Genstruktur aufwiesen. Beide ORFs lagen im Genom einander uberlappend vor, bildeten aber keinen einheitlichen Leserahmen. Demzufolge bestand das Enzym aus zwei Untereinheiten (50 kDa und 28 kDa) und zeigte eine heterooligomere Struktur. Die P. aerophilum ACD zeigte eine bisher fur ACD nicht beschriebene Substratspezifitat. Neben Acetyl-CoA wurden mit signifikanten Aktivitaten auch Isobutyryl-CoA und Phenylacetyl-CoA umgesetzt. Daneben wies das Enzym ein Temperaturoptimum von uber 93°C und hohe Thermostabilitat bis 100°C auf. Die von dem ORF PAE2867 kodierte ACS aus P. aerophilum ist ein thermostabiles Enzym mit einer fur ACS einzigartigen oligomeren Struktur. Das aus 77 kDa Untereinheiten bestehende Enzym wurde als 615 kDa Homooktamer charakterisiert. Das Enzym zeigte ein Temperaturoptimum von uber 97°C und wies eine breitere Substratspezifitat auf als alle bisher charakterisierten ACS. Neben Acetat wurden auch Formiat, Propionat, Butyrat und Isobutyrat als Substrate akzeptiert. Auserdem waren die Substrataffinitaten z.B. fur Acetat (Km-Wert 3 µM) 100fach hoher als fur die meisten charakterisierten ACS. Sequenzvergleiche der in dieser Arbeit charakterisierten archaeellen ACS mit anderen bekannten und putativen ACS Sequenzen zeigten, dass diese Enzyme in allen drei Domanen hochkonservierte Proteine sind. Phylogenetische Analysen mit ACS Sequenzen aus allen drei Domanen ergaben eine separate archaeelle Gruppe, zu der auch die ACS aus P.aerophilum gehorte. Dieses spricht fur eine monophyletische Entwicklungslinie der archaeellen ACS.
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identifizierung und charakterisierung der enzyme der acetatbildung und der acetataktivierung in halophilen und hyperthermophilen archaea adp bildende und amp bildende acetyl coa synthetasen aus Haloarcula marismortui und pyrobaculum aerophilum
2006Co-Authors: Christopher BrasenAbstract:Acetat ist eines der wichtigsten Endprodukte energieliefernder Fermentationsprozesse vieler anaerober oder fakultativer Bacteria und Archaea. Daruber hinaus stellt Acetat aber auch ein weit verbreitetes Substrat fur Katabolismus und Anabolismus aerober und anaerober, pro- und eukaryontischer Mikroorganismen dar. Bisherige Untersuchungen deuten darauf hin, dass der Mechanismus der Acetatbildung in Prokaryonten domanenspezifisch ist. In Bacteria wird die Acetatbildung von zwei Enzymen, der Acetat Kinase (AK) und der Phosphotransacetylase (PTA), katalysiert, wahrend in Archaea die Acetatbildung von nur einem Enzym, der ADP-bildende Acetyl-CoA Synthetase (ACD), erfolgt. Die Hauptmechanismen der Acetataktivierung, der weitverbreitete Mechanismus uber eine AMP-bildende Acetyl-CoA Synthetase (ACS) bzw. der von weniger Organismen beschrittene Mechanismus uber den reversiblen AK/PTA-Weg, kommen dagegen beide sowohl in Bacteria als auch in Archaea vor. Untersuchungen zur Acetatbildung in Archaea und dem daran beteiligten Enzym, der ACD, existierten bisher nur fur wenige hyperthermophile, anaerobe Euryarchaeota. Zudem lagen uber die Acetataktivierung in Archaea, auser fur die methanogenen Methanosaeta und Methanosarcina Spezies, keine Informationen vor. Daher sollten in dieser Arbeit das Wachstum der halophilen Archaea auf Glucose und Acetat untersucht und die Enzyme der Acetatbildung und -aktivierung identifiziert, charakterisiert sowie die kodierenden Gene ermittelt werden. Auserdem sollten die Gene, die fur die entsprechenden Enzyme in dem hyperthermophilen Crenarchaeon Pyrobaculum aerophilum kodieren, identifiziert, kloniert und exprimiert sowie die rekombinanten Proteine biochemisch charakterisiert werden. Identifizierung und Charakterisierung der Enzyme der Acetatbildung und -aktivierung in halophilen Archaea Wachstumsexperimente mit den extrem halophilen Archaea Haloarcula marismortui, Halorubrum saccharovorum, Haloferax volcanii und Halococcus saccharolyticus auf Glucose, Acetat und Glucose/Acetat enthaltenden Medien ergaben, dass die Haloarchaea Glucose und Acetat als Kohlenstoff- und Energiequelle nutzen konnen und dass Glucose gegenuber Acetat das bevorzugte Substrat ist. Zudem konnte mit Enzymmessungen in unterschiedlichen Wachstumsphasen gezeigt werden, dass die Aktivitaten von ACD und ACS substratabhangig reguliert werden. In Phasen der Acetatbildung bei Wachstum auf Glucose stieg die Aktivitat der ACD an, wohingegen die der ACS abnahm. Dagegen stieg in Phasen des Acetatverbrauches umgekehrt die ACS Aktivitat an, die der ACD nahm aber ab. Zusammen mit dem Befund, dass weder AK noch PTA nachgewiesen werden konnten, zeigten diese Experimente deutlich, dass die Acetatbildung in Haloarchaea von einer ACD katalysiert wird, wahrend die Acetataktivierung uber eine induzierbare ACS erfolgt. ACD und ACS wurden aus Ha. marismortui gereinigt und charakterisiert. Die kodierenden Gene wurden identifiziert, kloniert und die rekombinanten Proteine aus Einschlusskorpern reaktiviert und gereinigt. Die ACD ist ein 166 kDa Homodimer und katalysierte die reversible ADP- und Pi-abhangige Acetatbildung aus Acetyl-CoA. Das Enzym benotigte Hochsalzbedingungen (3 M KCl) fur die Stabilitat und setzte neben Acetat auch Propionat, Butyrat, Isobutyrat und Isovalerat, nicht aber die aromatischen Sauren Phenylacetat oder Indol-3-acetat um. Die ACS ist ein 71 kDa Monomer und katalysierte die ATP- und CoA abhangige Umsetzung von Acetat zu Acetyl-CoA, wobei AMP und PPi gebildet werden. Die ACS benotigte 1.25 M KCl fur optimale Aktivitat und setzte bevorzugt Acetat um und zusatzlich nur Propionat mit deutlich geringerer Effizienz. Der Km-Wert der ACS fur Acetat war mit 0.23 mM etwa 10fach kleiner als der der ACD (2.6 mM). Diese hohere Acetataffinitat konnte der Grund fur die Induktion der ACS bei Wachstum auf Acetat sein, weil Acetat v.a. in geringen Konzentrationen, wie sie in hypersalinen Habitaten vorkommen, erheblich effizienter aktiviert werden kann. ADP-bildende und AMP-bildende Acetyl-CoA Synthetasen aus dem hyperthermophilen Crenarchaeon Pyrobaculum aerophilum Im Genom von P. aerophilum wurden Gene identifiziert, die fur eine putative ACD und eine ACS kodieren. Die ORFs wurden exprimiert und die rekombinanten Proteine biochemisch charakterisiert. Es wurde gezeigt, dass die ORFs PAE3250 und PAE3249 aus P. aerophilum, die fur eine funktionelle ACD kodierten, eine fur bisher charakterisierte ACD ungewohnliche Genstruktur aufwiesen. Beide ORFs lagen im Genom einander uberlappend vor, bildeten aber keinen einheitlichen Leserahmen. Demzufolge bestand das Enzym aus zwei Untereinheiten (50 kDa und 28 kDa) und zeigte eine heterooligomere Struktur. Die P. aerophilum ACD zeigte eine bisher fur ACD nicht beschriebene Substratspezifitat. Neben Acetyl-CoA wurden mit signifikanten Aktivitaten auch Isobutyryl-CoA und Phenylacetyl-CoA umgesetzt. Daneben wies das Enzym ein Temperaturoptimum von uber 93°C und hohe Thermostabilitat bis 100°C auf. Die von dem ORF PAE2867 kodierte ACS aus P. aerophilum ist ein thermostabiles Enzym mit einer fur ACS einzigartigen oligomeren Struktur. Das aus 77 kDa Untereinheiten bestehende Enzym wurde als 615 kDa Homooktamer charakterisiert. Das Enzym zeigte ein Temperaturoptimum von uber 97°C und wies eine breitere Substratspezifitat auf als alle bisher charakterisierten ACS. Neben Acetat wurden auch Formiat, Propionat, Butyrat und Isobutyrat als Substrate akzeptiert. Auserdem waren die Substrataffinitaten z.B. fur Acetat (Km-Wert 3 µM) 100fach hoher als fur die meisten charakterisierten ACS. Sequenzvergleiche der in dieser Arbeit charakterisierten archaeellen ACS mit anderen bekannten und putativen ACS Sequenzen zeigten, dass diese Enzyme in allen drei Domanen hochkonservierte Proteine sind. Phylogenetische Analysen mit ACS Sequenzen aus allen drei Domanen ergaben eine separate archaeelle Gruppe, zu der auch die ACS aus P.aerophilum gehorte. Dieses spricht fur eine monophyletische Entwicklungslinie der archaeellen ACS.
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amp forming acetyl coa synthetase from the extremely halophilic archaeon Haloarcula marismortui purification identification and expression of the encoding gene and phylogenetic affiliation
Extremophiles, 2005Co-Authors: Christopher Brasen, Peter SchonheitAbstract:Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.
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regulation of acetate and acetyl coa converting enzymes during growth on acetate and or glucose in the halophilic archaeon Haloarcula marismortui
Fems Microbiology Letters, 2004Co-Authors: Christopher Brasen, Peter SchonheitAbstract:Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.
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unusual adp forming acetyl coenzyme a synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon pyrobaculum aerophilum
Archives of Microbiology, 2004Co-Authors: Christopher Brasen, Peter SchonheitAbstract:ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea (\({\text{Acetyl - CoA}} + {\text{ADP}} + {\text{P}}_{{\text{i}}} \rightleftarrows {\text{acetate}} + {\text{ATP}} + {\text{CoA}}\)), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for α and β subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits α (50 kDa) and β (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
Peter Schonheit - One of the best experts on this subject based on the ideXlab platform.
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amp forming acetyl coa synthetase from the extremely halophilic archaeon Haloarcula marismortui purification identification and expression of the encoding gene and phylogenetic affiliation
Extremophiles, 2005Co-Authors: Christopher Brasen, Peter SchonheitAbstract:Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.
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regulation of acetate and acetyl coa converting enzymes during growth on acetate and or glucose in the halophilic archaeon Haloarcula marismortui
Fems Microbiology Letters, 2004Co-Authors: Christopher Brasen, Peter SchonheitAbstract:Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.
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novel xylose dehydrogenase in the halophilic archaeon Haloarcula marismortui
Journal of Bacteriology, 2004Co-Authors: Ulrike Johnsen, Peter SchonheitAbstract:ABSTRACT During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui.
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unusual adp forming acetyl coenzyme a synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon pyrobaculum aerophilum
Archives of Microbiology, 2004Co-Authors: Christopher Brasen, Peter SchonheitAbstract:ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea (\({\text{Acetyl - CoA}} + {\text{ADP}} + {\text{P}}_{{\text{i}}} \rightleftarrows {\text{acetate}} + {\text{ATP}} + {\text{CoA}}\)), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for α and β subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits α (50 kDa) and β (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
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unusual adp forming acetyl coenzyme a synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon pyrobaculum aerophilum
Archives of Microbiology, 2004Co-Authors: Christopher Brasen, Peter SchonheitAbstract:ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea Acety - CoA + ADP + Pi acetate + ATP CoA), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for alpha and beta subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits alpha (50 kDa) and beta (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
Giuseppe Zaccai - One of the best experts on this subject based on the ideXlab platform.
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the oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies
Journal of Molecular Biology, 2003Co-Authors: Adriana Irimia, Christine Ebel, Giuseppe Zaccai, Dominique Madern, S B Richard, Lawrence W Cosenza, Frederic M D VellieuxAbstract:Abstract The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95 A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer–dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4 M NaCl, which dissociated into active dimers in 2 M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na 2 SO 4 , even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3 M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl − binding at the dimer–dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C α positions is 0.39 A). Chloride ions are also observed at the monomer–monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.
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characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui
Archaea, 2002Co-Authors: Bruno Franzetti, Guy Schoehn, D Garcia, Rob W H Ruigrok, Giuseppe ZaccaiAbstract:A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH) as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.
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characterization of a novel complex from halophilic archaebacteria which displays chaperone like activities in vitro
Journal of Biological Chemistry, 2001Co-Authors: Bruno Franzetti, Guy Schoehn, Christine Ebel, Jean Gagnon, Giuseppe ZaccaiAbstract:Abstract We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivoexperiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.
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halophilic adaptation novel solvent protein interactions observed in the 2 9 and 2 6 a resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui
Biochemistry, 2000Co-Authors: Stephane Richard, Dominique Madern, Elsa D Garcin, Giuseppe ZaccaiAbstract:Previous biophysical studies of tetrameric malate dehydrogenase from the halophilic archaeon Haloarcula marismortui (Hm MalDH) have revealed the importance of protein−solvent interactions for its adaptation to molar salt conditions that strongly affect protein solubility, stability, and activity, in general. The structures of the E267R stability mutant of apo (−NADH) Hm MalDH determined to 2.6 A resolution and of apo (−NADH) wild type Hm MalDH determined to 2.9 A resolution, presented here, highlight a variety of novel protein−solvent features involved in halophilic adaptation. The tetramer appears to be stabilized by ordered water molecule networks and intersubunit complex salt bridges “locked” in by bound solvent chloride and sodium ions. The E267R mutation points into a central ordered water cavity, disrupting protein−solvent interactions. The analysis of the crystal structures showed that halophilic adaptation is not aimed uniquely at “protecting” the enzyme from the extreme salt conditions, as may ha...
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insights into the molecular relationships between malate and lactate dehydrogenases structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric l ldh like malate dehydrogenase from the halophilic archaeon Haloarcula marismortui
Biochemistry, 2000Co-Authors: Dominique Madern, Christine Ebel, Moshe Mevarech, Claude Pfister, Stephane B Richard, Giuseppe ZaccaiAbstract:l-Malate (MalDH) and l-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dim...
Taketomo Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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expression purification crystallization and preliminary x ray analysis of the met244ala variant of catalase peroxidase katg from the haloarchaeon Haloarcula marismortui
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007Co-Authors: Tomomi Teni, Taketomo Fujiwara, Katsuhiko Yoshimatsu, Takashi Kumasaka, Wataru Higuchi, Satoru Tanaka, Takao SatoAbstract:The covalent modification of the side chains of Trp95, Tyr218 and Met244 within the active site of Haloarcula marismortui catalase-peroxidase (KatG) appears to be common to all KatGs and has been demonstrated to be particularly significant for its bifunctionality [Smulevich et al. (2006), J. Inorg. Biochem. 100, 568-585; Jakopitsch, Kolarich et al. (2003), FEBS Lett. 552, 135-140; Jakopitsch, Auer et al. (2003), J. Biol. Chem. 278, 20185-20191; Jakopitsch et al. (2004), J. Biol. Chem. 279, 46082-46095; Regelsberger et al. (2001), Biochem. Soc. Trans. 29, 99-105; Ghiladi, Knudsen et al. (2005), J. Biol. Chem. 280, 22651-22663; Ghiladi, Medzihradzky et al. (2005), Biochemistry, 44, 15093-15105]. The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant showed a complete loss of catalase activity, whereas the peroxidase activity of this mutant was highly enhanced owing to an increase in its affinity for the peroxidatic substrate. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 A, beta = 99.81 degrees . A crystal frozen using lithium sulfate as the cryoprotectant diffracted to beyond 2.0 A resolution. Preliminary X-ray analysis suggests the presence of a dimer in the asymmetric unit.
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Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon
Extremophiles, 2007Co-Authors: Katsuhiko Yoshimatsu, Osamu Araya, Taketomo FujiwaraAbstract:The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b- 561, cytochrome b- 552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b- 561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was −27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc . A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.
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The 2.0 Å crystal structure of catalase-peroxidase from Haloarcula marismortui
Nature structural biology, 2002Co-Authors: Yusuke Yamada, Taketomo Fujiwara, Takao Sato, Noriyuki Igarashi, Nobuo TanakaAbstract:Catalase-peroxidase is a member of the class I peroxidase superfamily. The enzyme exhibits both catalase and peroxidase activities to remove the harmful peroxide molecule from the living cell. The 2.0 A crystal structure of the catalase-peroxidase from Haloarcula marismortui (HmCP) reveals that the enzyme is a dimer of two identical subunits. Each subunit is composed of two structurally homologous domains with a topology similar to that of class I peroxidase. The active site of HmCP is in the N-terminal domain. Although the arrangement of the catalytic residues and the cofactor heme b in the active site is virtually identical to that of class I peroxidases, the heme moiety is buried inside the domain, similar to that in a typical catalase. In the vicinity of the active site, novel covalent bonds are formed among the side chains of three residues, including that of a tryptophan on the distal side of the heme. Together with the C-terminal domain, these covalent bonds fix two long loops on the surface of the enzyme that cover the substrate access channel to the active site. These features provide an explanation for the dual activities of this enzyme.
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sequence and electron paramagnetic resonance analyses of nitrate reductase nargh from a denitrifying halophilic euryarchaeote Haloarcula marismortui
FEBS Letters, 2002Co-Authors: Katsuhiko Yoshimatsu, Toshio Iwasaki, Taketomo FujiwaraAbstract:Genes encoding the NarG and NarH subunits of the molybdo–iron–sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe–4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe–4S] cluster and three [4Fe–4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain.
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purification characterization and genetic analysis of cu containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon Haloarcula marismortui
Journal of Bacteriology, 2001Co-Authors: Hirotaka Ichiki, Katsuhiko Yoshimatsu, Takeshi Sakurai, Yoko Tanaka, Kiyotaka Mochizuki, Taketomo FujiwaraAbstract:Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui . The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g II = 2.232; A II = 4.4 mT) and type 2 Cu centers (g II = 2.304; A II = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO 2 − · min −1 · mg −1 .
Moshe Mevarech - One of the best experts on this subject based on the ideXlab platform.
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insights into the molecular relationships between malate and lactate dehydrogenases structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric l ldh like malate dehydrogenase from the halophilic archaeon Haloarcula marismortui
Biochemistry, 2000Co-Authors: Dominique Madern, Christine Ebel, Moshe Mevarech, Claude Pfister, Stephane B Richard, Giuseppe ZaccaiAbstract:l-Malate (MalDH) and l-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dim...
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structural features that stabilize halophilic malate dehydrogenase from an archaebacterium
Science, 1995Co-Authors: Orly Dym, Moshe Mevarech, Joel L SussmanAbstract:The high-resolution structure of halophilic malate dehydrogenase (hMDH) from the archaebacterium Haloarcula marismortui was determined by x-ray crystallography. Comparison of the three-dimensional structures of hMDH and its nonhalophilic congeners reveals structural features that may promote the stability of hMDH at high salt concentrations. These features include an excess of acidic over basic residues distributed on the enzyme surface and more salt bridges present in hMDH compared with its nonhalophilic counterparts. Other features that contribute to the stabilization of thermophilic lactate dehydrogenase and thermophilic MDH—the incorporation of alanine into α helices and the introduction of negatively charged amino acids near their amino termini, both of which stabilize the α helix as a result of interaction with the positive part of the α-helix dipole—also were observed in hMDH.
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subunit structure of halophilic malate dehydrogenase from Haloarcula marismortui
Comparative Biochemistry and Physiology B, 1993Co-Authors: Ezra Daniel, Abdussalam Azem, Isabella Shaked, Moshe MevarechAbstract:Abstract 1. 1. The subunit structure of halophilic malate dehydrogenase from Haloarcula marismortui was studied by the method of crosslinking with bifunctional reagent. 2. 2. Exposure of the enzyme to glutardialdehyde followed by sodium dodecyl sulphate gel electrophoresis resulted in the appearance of four bands with mobilities corresponding to monomeric polypeptide chains and crosslinked polypeptide chain dimers, trimers and tetramers. 3. 3. Our findings are not consistent with the currently accepted dimeric structure of the enzyme. They provide a strong indication that halophilic malate dehydrogenase is composed of four identical subunits.
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cloning sequencing and expression in escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui
Biochemistry, 1993Co-Authors: Fabrice Cendrin, Giuseppe Zaccai, Jadwiga Chroboczek, Henryk Eisenberg, Moshe MevarechAbstract:The gene coding for the enzyme malate dehydrogenase (MDH) of the extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced. The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da. The deduced amino acid sequence of the enzyme was found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs. The structural gene was cloned in the Escherichia coli expression vector pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl. The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria. These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme. Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln. This modification produced an enzyme that has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH). The mutation also caused a modification in the relative activities of the enzyme at different salt concentrations. The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.
