Title page for ETD etd-10122005-134414


Type of Document Dissertation
Author Haddock, John David
URN etd-10122005-134414
Title Biochemistry and genetics of the pathway for the anaerobic degradation of aromatic compounds by Eubacterium oxidoreducens
Degree PhD
Department Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Ferry, James G. Committee Chair
Benoit, Robert E. Committee Member
Dean, Dennis R. Committee Member
Gregory, Eugene M. Committee Member
Wilkins, Tracy D. Committee Member
Keywords
  • Aromatic compounds Biodegradation.
Date of Defense 1990-08-05
Availability restricted
Abstract
The biochemical pathway for the anaerobic degradation of gallate, pyrogallol and phloroglucinol by Eubacterium oxidoreducens was investigated. Phloroglucinol reductase was purified 90-fold, from the soluble fraction of cell extract, to electrophoretic homogeneity. The enzyme was an α2 homodimer with a native Mr of 78,000, did not contain metals or cofactors and was specific for phloroglucinol and NADPH with a Km of 800 μM and 6.7 μM respectively at pH 6.8. The Km for phloroglucinol decreased with increasing pH. The enzyme catalyzed reaction was reversible and the equilibrium constant was 9.6. Dihydroresorcinol was a competitive inhibitor of the reverse reaction (Ki = 756 μM). Dihydrophloroglucinol produced in cell extract with H2 as the reductant was identical to the compound produced by sodium borohydride reduction of phloroglucinol as shown by 1H NMR spectroscopy. The 13C NMR spectrum was consistent with the structural assignment of dihydrophloroglucinol. The mechanism of the proposed enzymatically catalyzed reaction is proposed to involve transfer of a hydride equivalent from NADPH to the carbonyl carbon of the phloroglucinol dianion.

Mutant strains of E. oxidoreducens that showed no gallate decarboxylase or dihydrophloroglucinol hydrolase activity were isolated after mutagenesis with ethylmethane sulfonate and emichment with ampicillin. The decarboxylase deficient mutants were unable to grow on gallate while pyrogallol and phloroglucinol supported growth. The hydrolase deficient mutants were unable to grow on any aromatic substrates and converted gallate to pyrogallol and dihydrophloroglucinol. The conversion of gallate to non-aromatic intermediates by cell extract of the wild-type stain was dependent on the presence of 1,2,3,5-benzenetetrol for the conversion of pyrogallol to phloroglucinol and on formate for the reduction of phloroglucinol to dihydrophloroglucinol. Transhydroxylase activity involved in the conversion of pyrogallol to phloroglucinol was induced by growth on aromatic substrates. The formate dehydrogenase was located in the soluble fraction of cell extract, and activity was protected from oxygen inactivation by sodium azide. The Km for formate and NADP was 290 μM and 140 μM respectively at pH 7.5. The pH optimum for activity was 7.5 and maximum activity was observed at a temperature of 50° C.

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