Anaerober Toluol-Stoffwechsel in Thauera aromatica: Biochemische und spektroskopische Untersuchungen zur Reaktion der (R)-Benzylsuccinat Synthase
Der initiale Schritt des anaeroben Toluol-Abbaus in dem denitrifizierenden β-Proteobakterium Thauera aromatica wird durch eine stereospezifische Addition der Methylgruppe von Toluol an die Doppelbindung von Fumarat unter Bildung des Produkts (R)-Benzylsuccinat eingeleitet. Die Reaktion wird von der...
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Table of Contents: Anaerobic degradation of toluene in Thauera aromatica is initiated by stereospecific addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to form the first intermediate (R)-benzylsuccinate. This reaction is catalyzed by the enzyme (R)-benzylsuccinate synthase (BSS), which is a member of the glycyl-radical family together with the well characterized anaerobic ribonucleotide reductase, pyruvate formate lyase, glycerol dehydratase and 4-hydroxyphenylacetate decarboxylase. In contrast to other glycyl-radical enzymes BSS consists of three different subunits (α2β2γ2-composition) and contains iron-sulfur cofactors with so far unknown function. All members of the glycyl-radical family can be identified on the basis of the characteristic glycyl-radical signal in EPR-spectroscopic analysis. For catalytic activity, glycyl-radical enzymes require a posttranslational activation by dedicated activating enzymes. All enzymes involved in the degradation of toluene to (R)-benzylsuccinate are encoded in the toluene-inducible bss-operon. I The FeS-cofactors of the recombinant non-activated BSS were characterized by UV/vis-, EPR- and Mössbauer-spectroscopy. With these methods at least two different types of magnetically coupled [4Fe4S]-clusters per holoenzyme were identified. The redox potential of the FeS-clusters seems to be lower than – 450 mV and the magnetic coupling suggests a close distance of the clusters relative to each other (< 10 Å). Based on aminoacid sequence alignments of other fumarate adding enzymes, the clusters are predicted to be localized in the small β- and γ- subunits of BSS. Furthermore recombinant BSS was used for crystallization experiments yielding a resolution of 4 Å. The achieved resolution is not sufficient for solving the crystal structure in detail, but the cell content analysis confirmed the postulated α2β2γ2-composition of BSS. II We also investigate the reaction mechanism of (R)-benzylsuccinate synthase by biochemical and EPR-spectroscopic studies of native BSS. The significant reduction of the reaction rate in enzyme assays with [2H2]toluene implies, that the abstraction of the hydrogen atom from toluene is indeed the rate limiting step of the BSS-reaction, as expected from the theoretical prediction. Detailed kinetic studies were achieved with substrate- and product-related inhibitors. The received kinetic parameters support the postulated BSS reaction mechanism. Identification and characterization of radical intermediates in the BSS-reaction are extremely helpful to investigate the reaction mechanism. During the time-course of this work a first direct observation of an organic radical intermediate in a glycyl radical enzyme was accomplished by EPR-spectroscopy. The results support significantly the proposed reaction mechanism of BSS and in general the assumed mechanisms via radical intermediates of all glycyl radical enzymes. III The activating enzyme (BssD) was recombinantly produced in E. coli and purified by His-Tag affinity-chromatography under strict anaerobic conditions. The enzyme was characterized by UV-Vis spectroscopy, EPR spectroscopy and chemical analysis and contains redox-active [4Fe4S]-clusters as predicted from aminoacid sequence alignments. Based on these results, further studies on the activation reaction of BSS can be accomplished.