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Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus

Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus

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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 Konstanz

@phdthesis{Niessen2004Struc-7932, title={Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus}, year={2004}, author={Niessen, Holger}, address={Konstanz}, school={Universität Konstanz} }

2011-03-24T17:38:37Z deposit-license Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus 2004 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.<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. Niessen, Holger 2011-03-24T17:38:37Z application/pdf Niessen, Holger deu Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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