Publikation: Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus
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Zusammenfassung
P. acetylenicus is a strictly anaerobic and mesophilic bacterium that is able to grow on acetylene as single energy and carbon source. The first step in the metabolization of acetylene is the transformation of acetylene to acetaldehyde. This addition of water is catalyzed by the W/Fe-S dependent enzyme acetylene hydratase.
Acetylene hydratase from P. acetylenicus was purified to homogeneity. It is a monomer with a molecular mass of the amino acid chain of 81.9 kDa. BLASTP searches revealed that the enzyme is highly similar to enzymes of the DMSO-reductase family. Acetylene hydratase is a thermostable enzyme with a temperature optimum between 50 and 55°C. It is a very stable enzyme when stored under exclusion of dioxygen in a nitrogen/hydrogen atmosphere at 6°C (Abt, 2001). Within three months, there was no detectable loss of acetylene hydratase activity from tungstate-grown P. acetylenicus. Although acetylene hydratase catalyzes no redox reaction, it contains one [4Fe-4S] center and one W-bisMGD as redox-cofactors.
Crystals of the w-acetylene hydratase were obtained both in presence and in absence (N2: H2 = 94: 6 v/v) of dioxygen. Only the crystals grown in absence of dioxygen were able to diffract X-ray-radiation. Dithionite-reduced enzyme crystals obtained under exclusion of dioxygen, could be measured at the Deutsches Elektronensynchrotron (DESY) in Hamburg up to resolution better then 2,5Å. Also crystals were measured at the ESRF in Grenoble at the tungsten L-edge, however, this dataset could not be used to solve the three dimensional structure because of high mosaicity and decreasing of resolution. This crystal was not stably enough to stand the measurement in the synchrotron radiation.
P. acidigallici is a strictly anaerobic bacterium that is able to live on gallic acid (3,4,5-trihydroxybenzoic acid), pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxy-benzene), or 2,4,6-trihydroxybenzoic acid. A crucial step in the fermentation of decarboxylated gallic acid (pyrogallol) is the transhydroxylation of pyrogallol to phloroglucinol. This reaction is catalyzed by the Mo/Fe-S dependent enzyme transhydroxylase (pyrogallol:phloroglucinol hydroxyltransferase E.C. 1.97.1.2).
Transhydroxylase from P. acidigallici is a heterodimer consisting of a large subunit (100.4 kDa) and a small subunit (31.3 kDa). This enzyme is closely related to enzymes of the DMSO-reductase family. Although the overall reaction of transhydroxylase is no redox reaction it contains different iron-sulfur centers and one Mo-bisMGD as redox-cofactors.
12 of the 13 cysteines in the small β-subunit are highly conserved. Some of them are referred to the [4Fe-4S] ferredoxins. The 15 cysteines of the big subunit do not align with the cysteines of related iron-sulfur proteins. Therefore, it is unlikely that an iron-sulfur center is located in the large subunit. It is more likely that there are three [4Fe-4S] clusters located in the small subunit.
Crystals of as isolated transhydroxylase were not able to diffract X-ray radiation. Crystallization of dithionite reduced transhydroxylase under exclusion of dioxygen led to crystals which diffracted to resolution limits higher than 2.5 Å with synchrotron radiation.
These crystals were measured with synchrotron radiation and the three-dimensional structure of transhydroxylase was solved. Even structures of transhydroxylase in complex with pyrogallol and inhibitor (1,2,4-trihydroxybenzene) were solved at high resolutions up to 2.0 Å.
These result led to a new possible reaction mechanism. Hereby the function of the molybdenum ion is to coordinate the pyrogallol at its C1 position. The amino acids Asp A174, His A144 and Tyr A404 near the active site seem to have catalytic function.
Zusammenfassung in einer weiteren Sprache
P. acetylenicus ist ein mesophiles, strikt anaerob lebendes Bakterium, das in der Lage ist, auf Acetylen als einziger Kohlenstoff- und Energiequelle zu wachsen. Die Metabolisierung von Acetylen wird durch das W/Fe-S abhängige Enzym Acetylenhydratase eingeleitet, wobei in einer ungewöhnlichen Reaktion Acetylen zu Acetaldehyd hydratisiert wird.
Das Enzym Acetylenhydratase wurde aus P. acetylenicus zur Homogenität gereinigt. Es handelt sich um ein Monomer mit einer molekularen Masse der Aminosäurekette von 81.9 kDa. Das Enzym gehört zur Familie der DMSO-Reduktasen. Acetylenhydratase ist ein thermostabiles Enzym, dessen Temperaturoptimum im Bereich von 50 bis 55°C liegt. In einer Stick-stoff/Wasserstoff Atmosphäre bei 6°C konnte das Enzym 3 Monate gelagert werden, ohne daß ein Aktivitätsverlust festgestellt wurde (Abt, 2001). Obwohl die Acetylenhydratase keine Redox-Reaktion katalysiert, enthält sie ein [4Fe-4S] Zentrum und einen W-bisMGD Kofaktor.
Kristalle der W-Acetylenhydratase wurden in Anwesenheit und in Abwesenheit (N2 : H2 = 94 : 6 v/v) von Luftsauerstoff erhalten. Jedoch nur die in Abwesenheit von Luftsauerstoff erhaltenen Kristalle waren in der Lage Röntgenstrahlen zu beugen. Dithionit-reduziertes Enzym ergab unter Ausschluß von Luftsauerstoff Kristalle, die am Deutschen Elektronen Synchrotron (DESY) in Hamburg vermessen wurden und bis zu einer Auflösung besser als 2,5 Å streuten. Ebenfalls wurden Kristalle am ESRF in Grenoble an der Wolfram L-Kante vermessen, jedoch konnte dieser Datensatz aufgrund sehr hoher Mosaizität und einer Abnahme der Auflösung nicht benutzt werden die 3-dimensionale Struktur zu ermitteln. Dieser Kristall war nicht stabil genug der Synchrotronstrahlung standzuhalten.
P. acidigallici ist ein strikt anaerob lebendes Bakterium, das in der Lage ist, mit Gallussäure (3,4,5-Trihydroxybenzoesäure), Pyrogallol (1,2,3-Trihydroxybenzol), Phloroglucin (1,3,5-Tri-hydroxybenzol) oder 2,4,6-Trihydroxybenzoesäure als einziger Kohlenstoff- und Energiequelle zu leben. Ein entscheidender Schritt während der Metabolisierung von decarboxylierter Gallussäure (Pyrogallol) ist die Transhydroxylierung des Pyrogallols zum Phloroglucin. Diese Reaktion wird von dem Mo/Fe-S abhängigen Enzym Transhydroxylase (Pyrogallol:Phloroglucin Hydroxyltransferase E.C. 1.97.1.2) katalysiert.
Es handelt sich um ein Heterodimer, das aus einer großen Untereinheit (100,4 kDa) und einer kleinen Untereinheit (31,3 kDa) besteht. Das Enzym ist eng mit Mitgliedern der DMSO-Reduktase Familie verwandt. Obwohl die Gesamtreaktion der Transhydroxylase keine Redoxreaktion ist, enthält das Enzym einen Mo-bisMGD Redoxkofaktor und verschiedene Eisen-Schwefel Zentren.
12 der 13 Cysteine der kleinen Untereinheit der Transhydroxylase sind hochkonserviert. Einige davon sind als [4Fe-4S] Ferredoxine beschrieben worden. Die 15 Cysteine der großen Untereinheit lassen sich nicht mit den Cysteinen anderer Proteine abgleichen. Aus diesem Grund ist es wahrscheinlich, daß die Eisen-Schwefel Zentren sich in der kleinen Untereinheit befinden.
Experimente mit as isolated -Transhydroxylase führten zu Kristallen, die im Röntgenstrahl nicht beugten. Die Kristallisation von Dithionit-reduzierter Transhydroxylase unter anoxischen Bedingungen in einer Stickstoff/Wasserstoff-Atmosphäre führte zu Kristallen, die mit Synchrotonstrahlung eine Auflösung von mehr als 2,5 Å ereichten.
Diese Kristalle konnten mit Synchrotronstrahlung vermessen und die 3-dimensional Struktur der Transhydroxylase ermittelt werden. Ebenfalls konnten hochaufgelöste Strukturen der Transhydroxylase mit Pyrogallol und Inhibitor (1,2,4-Trihydroxybenzol) mit einer Auflösung von bis zu 2.0 Å ermittelt werden. Diese Ergebnisse ließen Schlussfolgerungen über einen neuartigen möglichen Reaktionsmechanismus zu. In diesem Falle hat das Molybdän die Funktion das Pyrogallol an dessen C1 Position zu koordinieren. Katalytische Funktionen haben hierbei die Aminosäuren Asp A174, His A144 und Tyr A404 in der unmittelbaren Nähe zum aktiven Zentrum.
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NIESSEN, Holger, 2004. Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus [Dissertation]. Konstanz: University of KonstanzBibTex
@phdthesis{Niessen2004Struc-7932, year={2004}, title={Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus}, author={Niessen, Holger}, address={Konstanz}, school={Universität Konstanz} }
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The first step in the metabolization of acetylene is the transformation of acetylene to acetaldehyde. This addition of water is catalyzed by the W/Fe-S dependent enzyme acetylene hydratase.<br />Acetylene hydratase from P. acetylenicus was purified to homogeneity. It is a monomer with a molecular mass of the amino acid chain of 81.9 kDa. BLASTP searches revealed that the enzyme is highly similar to enzymes of the DMSO-reductase family. Acetylene hydratase is a thermostable enzyme with a temperature optimum between 50 and 55°C. It is a very stable enzyme when stored under exclusion of dioxygen in a nitrogen/hydrogen atmosphere at 6°C (Abt, 2001). Within three months, there was no detectable loss of acetylene hydratase activity from tungstate-grown P. acetylenicus. Although acetylene hydratase catalyzes no redox reaction, it contains one [4Fe-4S] center and one W-bisMGD as redox-cofactors.<br />Crystals of the w-acetylene hydratase were obtained both in presence and in absence (N2: H2 = 94: 6 v/v) of dioxygen. Only the crystals grown in absence of dioxygen were able to diffract X-ray-radiation. Dithionite-reduced enzyme crystals obtained under exclusion of dioxygen, could be measured at the Deutsches Elektronensynchrotron (DESY) in Hamburg up to resolution better then 2,5Å. Also crystals were measured at the ESRF in Grenoble at the tungsten L-edge, however, this dataset could not be used to solve the three dimensional structure because of high mosaicity and decreasing of resolution. This crystal was not stably enough to stand the measurement in the synchrotron radiation.<br /><br />P. acidigallici is a strictly anaerobic bacterium that is able to live on gallic acid (3,4,5-trihydroxybenzoic acid), pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxy-benzene), or 2,4,6-trihydroxybenzoic acid. A crucial step in the fermentation of decarboxylated gallic acid (pyrogallol) is the transhydroxylation of pyrogallol to phloroglucinol. This reaction is catalyzed by the Mo/Fe-S dependent enzyme transhydroxylase (pyrogallol:phloroglucinol hydroxyltransferase E.C. 1.97.1.2).<br />Transhydroxylase from P. acidigallici is a heterodimer consisting of a large subunit (100.4 kDa) and a small subunit (31.3 kDa). This enzyme is closely related to enzymes of the DMSO-reductase family. Although the overall reaction of transhydroxylase is no redox reaction it contains different iron-sulfur centers and one Mo-bisMGD as redox-cofactors.<br />12 of the 13 cysteines in the small β-subunit are highly conserved. Some of them are referred to the [4Fe-4S] ferredoxins. The 15 cysteines of the big subunit do not align with the cysteines of related iron-sulfur proteins. Therefore, it is unlikely that an iron-sulfur center is located in the large subunit. It is more likely that there are three [4Fe-4S] clusters located in the small subunit.<br />Crystals of as isolated transhydroxylase were not able to diffract X-ray radiation. Crystallization of dithionite reduced transhydroxylase under exclusion of dioxygen led to crystals which diffracted to resolution limits higher than 2.5 Å with synchrotron radiation.<br />These crystals were measured with synchrotron radiation and the three-dimensional structure of transhydroxylase was solved. Even structures of transhydroxylase in complex with pyrogallol and inhibitor (1,2,4-trihydroxybenzene) were solved at high resolutions up to 2.0 Å.<br />These result led to a new possible reaction mechanism. Hereby the function of the molybdenum ion is to coordinate the pyrogallol at its C1 position. The amino acids Asp A174, His A144 and Tyr A404 near the active site seem to have catalytic function.</dcterms:abstract> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7932"/> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7932/1/DissertationHolgerNiessen.pdf"/> <dc:contributor>Niessen, Holger</dc:contributor> <dc:rights>terms-of-use</dc:rights> <foaf:homepage rdf:resource="http://localhost:8080/"/> <dcterms:issued>2004</dcterms:issued> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> </rdf:Description> </rdf:RDF>