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Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by Azoarcus anaerobius : biochemical aspects of the degradation pathway and identification of involved genes

Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by Azoarcus anaerobius : biochemical aspects of the degradation pathway and identification of involved genes

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HELLSTERN, Jutta A., 2005. Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by Azoarcus anaerobius : biochemical aspects of the degradation pathway and identification of involved genes [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Hellstern2005Anaer-7754, title={Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by Azoarcus anaerobius : biochemical aspects of the degradation pathway and identification of involved genes}, year={2005}, author={Hellstern, Jutta A.}, address={Konstanz}, school={Universität Konstanz} }

2005 Hellstern, Jutta A. eng 2011-03-24T17:37:17Z Hellstern, Jutta A. The present thesis deals with investigations on an hitherto unknown pathway of resorcinol (1,3-dihydroxybenzene) degradation by the nitrate-reducing bacterium Azoarcus anaerobius.<br />Resorcinol is oxidized by membrane-bound enzymes via hydroxyhydroquinone (1,2,4-trihydroxybenzene, HHQ) to 2-hydroxy-1, 4-benzoquinone (hydroxybenzoquinone, HBQ). In the focus of this thesis is the further degradation of HBQ and the identification of genes involved in resorcinol degradation.<br />Degradation of HBQ was investigated in cell-free extracts of A. anaerobius. Resorcinol was converted into stoichiometric amounts of acetate, malate, and succinate in the presence of nitrate and NADH. During resorcinol degradation, unstable aldehydes and ketones were trapped as intermediates by derivatization with 2,4-dinitrophenylhydrazine. The conversion of HBQ to acetate, malate, and succinate was catalyzed only by cell-free extracts of resorcinol-grown cells.<br />By heterologous expression of a gene library containing fragments of the A. anaerobius genome, a genome fragment R+ was obtained which enabled two Thauera aromatica strains, AR-1 and K 172, to grow with resorcinol as sole carbon and energy source plus nitrate as electron acceptor. The genome fragment R+ was 29.88 kb in size. Physiological and biochemical experiments showed that R+ must harbour genes for resorcinol hydroxylase and HHQ dehydrogenase, and genes for the conversion of HBQ to acetate, malate, and succinate. Genes involved in resorcinol metabolism including the specific regulatory units were assigned to four adjacent gene clusters.<br />Growth experiments and enzyme activity tests with inhibitors for molybdenum-containing enzymes (arsenite, tungstate, selenite) suggested that a molybdenum-containing enzyme catalysed the hydroxylation of resorcinol to HHQ. Specific motifs of molybdenum-containing enzymes (molybdenum and iron-sulfur binding sites) were found in the genome fragment R+. The respective ORFs, assigned as putative resorcinol hydroxylase, showed identities of ~50% to the a- and b-subunits of the pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici.<br />Sequences of oligopeptides of a 50 kDa protein from membrane fractions of A. anaerobius grown with resorcinol aligned to the predicted HHQ dehydrogenase in the genome fragment R+. The 50 kDa protein was present in membrane fractions of A. anaerobius, wild-type T. aromatica AR-1 (wt), and the two transconjugantes T. aromatica AR-1 R+ and K 172 R+, independent of the used growth substrate. The intensity of this protein band correlated with the specific activity of HHQ dehydrogenase in the respective cell-free extracts. The observation that the intensity of the 50 kDa protein band and the specific activity of HHQ dehydrogenase both were substantially higher in trans-conjugate T. aromatica AR-1 R+ than in wild-type T. aromatica AR-1 during growth on resorcinol or a-resorcylate suggested that trans-conjugate T. aromatica AR-1 R+ possesses two gene copies for the HHQ dehydrogenase.<br />The genes for HBQ ring cleavage were assigned to the ORFs that resembled the multi-enzyme complex of pyruvate dehydrogenase on the genome fragment R+. Typical binding-motifs for the co-enzymes and -substrates of pyruvate dehydrogenase (CoA, FAD, NAD+, TPP) were localized. However, addition of these co-enzymes and/or -substrates in cell-free extract experiments did not support resorcinol degradation. HBQ was cleaved most likely in a non-oxidative hydrolytic reaction to a b-ketoacid, which was subsequently cleaved to a C2 and a C4 moiety.<br />The genes for the subsequent reactions to form acetate, malate, and finally succinate were assigned to putative NADH-dependent, soluble dehydrogenases. These enzymes transformed the C2 moiety to acetate and the C4 moiety via malate to succinate. Anaerobic degradation of resorcinol (1,3-dihydroxybenzene) by Azoarcus anaerobius : biochemical aspects of the degradation pathway and identification of involved genes application/pdf 2011-03-24T17:37:17Z deposit-license Anaerober Abbau von Resorcin (1,3- Dihydroxybenzol) durch Azoarcus anaerobius: biochemische Aspekte des Abbauweges und Identifizierung der beteiligten Gene

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