Characterization of the degradation pathway of phloroglucinol (1,3,5-trihydroxybenzene) by a Rhodococcus sp. BPG-8

Armstrong, Stephen Mark (1992) Characterization of the degradation pathway of phloroglucinol (1,3,5-trihydroxybenzene) by a Rhodococcus sp. BPG-8. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Rhodococcus sp. BPG-8 was isolated from oil rich soil in Newfoundland and found to utilize numerous aromatic compounds as sources of carbon and energy. Chemical analysis of cell wall composition which included amino acids, sugars, and fatty acids showed complete homology with Rhodococcus erythropolis. Identical profiles for acid production and growth on various substrates occurred. Growth of the isolate on phloroglucinol occurred in the pH range 5-8; with substrate and temperature optima of 8.0 mM and 25°C, respectively. Phloroglucinol induced cells when fed phloroglucinol or resorcinol produced 1,2,3,5-tetrahydroxybenzene and 1,2,4-trihydroxybenzene, respectively. Cell-free extracts of cells grown on phloroglucinol contained a phloroglucinol hydroxylase that also hydroxylated resorcinol. Dioxygenases present in the induced cells carried out the ortho-cleavage of 1,2,3,5- tetrahydroxybenzene while meta-clevage of 1,2,4- trihydroxybenzene appeared to be constitutive. Cell-free extracts also showed inducible activity for the metabolism of acetopyruvate with the accumulation of formate in the supernatant. Tentative degradative pathways for phloroglucinol and fortuitous resorcinol metabolism are discussed. This is the first reported case in which phloroglucinol is metabolized by an oxidative rather than a reductive pathway. The oxidations of 1,2,3,5-tetrahydroxybenzene and 1,2,4- trihydroxybenzene produce superoxide radicals that may have potential deleterious effects on cellular integrity. It has been shown that both superoxide dismutase and catalase retard the auto-oxidation of these molecules by hindering their free radical reaction mechanism with superoxide. A non-inducible NAD(P)H dependent reductase appeared to convert the 2-hydroxy- 1,4-benzoquinone back to 1,2,4-trihydroxybenzene; although similiar effects were not found for 1,2,3,5- tetahydroxybenzene. These novel findings suggest that constitutive non-pathway enzymes may participate in stabilization of intermediates. Partial purification of the phloroglucinol hydroxylase was performed using ammonium sulfate percipitation, ion exchange chromatography, and gel filtration. The pH, temperature, and substrate optima for phloroglucinol hydroxylase were 7.0, 25°C, and 68.0 μM for the substrates phloroglucinol and resorcinol. NADH+H was the primary reductant and FAD stimulated the hydroxylase activity by 300 %. The enzyme had a native molecular weight of 155,000 daltons and an apparent Km of 8.3 μM and 12.5 μM for phlorglucinol and resorcinol respectively. Chloride ion along with numerous metal ions appeared to inhibit phloroglucinol and resorcinol hydroxylase activities. This is the first reported case for the partial purification of a phloroglucinol hydroxylase.

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