Abstract
Anaerobic degradation of (4-hydroxy)phenylacetate in denitrifying Pseudomonas sp. was investigated. Evidence is presented for α-oxidation of the coenzyme A (CoA)-activated carboxymethyl side chain, a reaction which has not been described. The C6−C2 compounds are degraded to benzoyl-CoA and furtheron to CO2 via the following intermediates: Phenylacetyl-CoA, phenylglyoxylate, benzoyl-CoA plus CO2; 4-hydroxyphenylacetyl-CoA, 4-hydroxyphenylglyoxylate, 4-hydroxybenzoyl-CoA plus CO2, benzoyl-CoA. Trace amounts of mandelate possibly derived from mandelyl-CoA were detected during phenylacetate degradation in vitro. The reactions are catalyzed by (i) phenylacetate-CoA ligase which converts phenylacetate to phenylacetyl-CoA and by a second enzyme for 4-hydroxyphenylacetate; (ii) a (4-hydroxy)-phenylacetyl-CoA dehydrogenase system which oxidizes phenylacetyl-CoA to (4-hydroxy)phenylglyoxylate plus CoA; and (iii) (4-hydroxy)phenylglyoxylate: acceptor oxidoreductase (CoA acylating) which catalyzes the oxidative decarboxylation of (4-hydroxy)phenylglyoxylate to (4-hydroxy)benzoyl-CoA and CO2. (iv) The degradation of 4-hydroxyphenylacetate in addition requires the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, catalyzed by 4-hydroxybenzoyl-CoA reductase (dehydroxylating). The whole cell regulation of these enzyme activities supports the proposed pathway. An ionic mechanism for anaerobic α-oxidation of the CoA-activated carboxymethyl side chain is proposed. Phenylacetic acids are plant constituents and in addition are formed from a large variety of natural aromatic compounds by microorganisms; their degradation therefore plays a significant role in nature, as illustrated in the preceding paper (Mohamed and Fuchs 1993). We have investigated and purified an enzyme which catalyzes the first step in the anaerobic degradation of phenylacetate in a denitrifying Pseudomonas sp. Phenylacetate is converted to phenylacetyl-CoA by phenylacetate-CoA ligase (AMP forming). The postulated function of this enzyme is corroborated by the strict regulation of its expression. 4-Hydroxyphenylacetate appears to be similarly activated by an independent enzyme prior to further degradation.
We have suggested before that phenylacetyl-CoA is anaerobically converted by α-oxidation of the side chain to phenylglyoxylate1, which is oxidatively decarboxylated to benzoyl-CoA plus CO2 (Seyfried et al. 1991; Dangel et al. 1991). 4-Hydroxyphenylacetate was proposed to be similarly oxidized to 4-hydroxybenzoyl-CoA plus CO2, followed by reductive dehydroxylation to benzoyl-CoA. The evidence was not presented in full, and the crucial α-oxidation was not demonstrated in vitro. We present here ample evidence for this pathway. A hypothetical mechanism is proposed by which the oxidation of the α-methylene group to an α-carbonyl group may occur.
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Mohamed, M.ES., Seyfried, B., Tschech, A. et al. Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomonas sp.. Arch. Microbiol. 159, 563–573 (1993). https://doi.org/10.1007/BF00249036
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DOI: https://doi.org/10.1007/BF00249036