Archives of Microbiology

, Volume 159, Issue 6, pp 563–573 | Cite as

Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomonas sp.

Evidence for an α-oxidation mechanism
  • Magdy El-Said Mohamed
  • Birgit Seyfried
  • Andreas Tschech
  • Georg Fuchs
Original Papers


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.

Key words

Phenylacetate 4-Hydroxyphenylacetate Phenylglyoxylate Alpha-Oxidation Pseudomonas Oxidoreductase CoA ligase Benzoyl-CoA Anaerobic aromatic metabolism 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bak F, Widdel F (1986) Anaerobic degradation of phenol and phenol derivatives by Desulfobacterium phenolicum sp. nov. Arch Microbiol 146: 177–180CrossRefGoogle Scholar
  2. Balba MT, Evans WC (1979) The methanogenic fermentation of omega-phenylalkane carboxylic acids. Biochem Soc Trans 7: 403–405CrossRefGoogle Scholar
  3. Blakley ER, Kurz W, Halvorson H, Simpson FJ (1967) The metabolism of phenlacetic acid by a Pseudomonas. Can J Microbiol 13: 147–157CrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254CrossRefGoogle Scholar
  5. Braun K, Gibson DT (1984) Anaerobic degradation of 2-aminobenzoate (anthranilic acid) by denitrifying bacteria. Appl Environ Microbiol 48: 102–107PubMedPubMedCentralGoogle Scholar
  6. Buckel W (1990a) Amino acid fermentation: Coenzyme B12-dependent and-independent pathways. In: Hauska G, Thauer R (eds) The molecular basis of bacterial metabolism. Springer, Berlin Heidelberg New York, pp 21–30CrossRefGoogle Scholar
  7. Buckel W (1990b) Unusual dehydrations in anaerobic bacteria. FEMS Microbiol Rev 88: 211–232CrossRefGoogle Scholar
  8. Cooper RA, Skinner MA (1980) Catabolism of 3- and 4-hydroxyphenylacetate by the 3,4-dihydroxyphenylacetate pathway in Escherichia coli. J Bacteriol 143: 302–306PubMedPubMedCentralGoogle Scholar
  9. D'Ari L, Barker HA (1985) p-Cresol formation by cell-free extracts of Clostridium difficile. Arch Microbiol 143: 311–312CrossRefGoogle Scholar
  10. Dangel W, Brackmann R, Lack A, Magdy M, Koch J, Oswald B, Seyfried B, Tschech A, Fuchs G (1991) Differential expression of enzymes initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA. Arch Microbiol 155: 256–262CrossRefGoogle Scholar
  11. Decker K (1959) Die aktivierte Essigsäure. Enke, StuttgartGoogle Scholar
  12. Dörner C, Schink B (1991) Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium. Arch Microbiol 156: 302–306CrossRefGoogle Scholar
  13. Fewson CA (1988) Microbial metabolism of mandelate: a microcosm of diversity. FEMS Microbiol Rev 54: 85–110CrossRefGoogle Scholar
  14. Gesellschaft Deutscher Chemiker (1979) Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung, 3rd edn. Verlag Chemie, WeinheimGoogle Scholar
  15. Glöckler R, Tschech A, Fuchs G (1989) Reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA in a denitrifying, phenol degrading Pseudomonas species. FEBS Lett 251: 237–240CrossRefGoogle Scholar
  16. Grbic-Galic D (1985) Fermentative and oxidative transformation of ferulate by a facultatively anaerobic bacterium isolated from sewage sludge. Appl Environ Microbiol 50: 1052–1057PubMedPubMedCentralGoogle Scholar
  17. Grbic-Galic D (1986) Anaerobic production and transformation of aromatic hydrocarbons and substituted phenols by ferulic acid-degrading BESA-inhibited methanogenic consortia. FEMS Microbiol Ecol 38: 161–169CrossRefGoogle Scholar
  18. Härtel U, Eckel E, Koch J, Fuchs G, Linder D, Buckel W (1993) Purification of glutaryl-CoA dehydrogenase from Pseudomnas sp., an enzyme involved in the anaerobic degradation of benzoate. Arch Microbiol 259: 174–181CrossRefGoogle Scholar
  19. Healy JB, Young LY, Reinhard M (1980) Methanogenic decomposition of perulic acid, a model lignin derivative. Appl Environ Microbiol 39: 436–444PubMedPubMedCentralGoogle Scholar
  20. Hockenhull DJD, Walker AD, Wilkin GD, Winder FG (1952) Oxidation of phenylacetic acid by Penicillium chrysogenum. Biochem J 50: 605–609CrossRefGoogle Scholar
  21. Kerscher L, Oesterhelt D (1982) Pyruvate: feredoxin oxidoreductase — new findings on an ancient enzyme. TIBS 7: 371–374Google Scholar
  22. Kishore G, Sugumaran M, Vaidyanathan CS (1976) Metabolism of dl-(±)phenylalanine by Aspergillus niger. J Bacteriol 128: 182–191PubMedPubMedCentralGoogle Scholar
  23. Koch J, Fuchs G (1992) Enzymatic reduction of benzoyl-CoA to alicyclic compounds, a key reaction in anaerobic aromatic metabolism. Eur J Biochem 205: 195–202CrossRefGoogle Scholar
  24. Koch J, Eisenreich W, Bacher A, Fuchs G (1993) Products of enzymatic reduction of benzoyl-CoA, a key reaction in anaerobic aromatic metabolism. Eur J Biochem 211: 649–661CrossRefGoogle Scholar
  25. Kuchta RD, Hanson GR, Homquist B, Abeles RH (1986) Fe−S centers in lactyl-CoA dehydratase. Biochemistry 25: 7301–7307CrossRefGoogle Scholar
  26. Kurz LC, Saurabh S, Crane BR, Donald LJ, Duckworth HW, Drysdale GR (1992) Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction. Biochemistry 31: 7899–7907CrossRefGoogle Scholar
  27. Lack A, Tommasi I, Aresta M, Fuchs G (1991) Catalytic properties of phenol carboxylase: in vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction. Eur J Biochem 197: 473–479CrossRefGoogle Scholar
  28. Landymore AF, Antia NJ, Towers GHN (1978) The catabolism of l-phenylalanine, l-tyrosine, and some related aromatic compounds by two marine species of phytoplankton. Phycologia 17: 319–328CrossRefGoogle Scholar
  29. Madigan MT, Gest H (1988) Selective enrichment and isolation of Rhodopseudomonas palustris using transcinnamic acid as sole carbon source. FEMS Microbiol Ecol 53: 53–58CrossRefGoogle Scholar
  30. Manley SL, Chapman DJ (1979) Metabolism of l-tyrosine to 4-hydroxybenzaldehyde and 3-bromo-4-hydroxybenzaldehyde by chloroplast-containing fractions of Odonthalia floccosa (Esp.) Falk. Plant Physiol 64: 1032–1038CrossRefGoogle Scholar
  31. Martin M, Gibello A, Fernandez J, Ferrer E, Garrido-Pertierra A (1991) Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae. J Gen Microbiol 132: 621–628CrossRefGoogle Scholar
  32. Martinez-Blanco H, Reglero A, Rodriguez-Aparicio LB, Luengo JM (1990) Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid. J Biol Chem 265: 7084–7090PubMedGoogle Scholar
  33. Mohamed M, Fuchs G (1993) Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate. Arch Microbiol 159: 561–569Google Scholar
  34. Perrin PW, Towers CHN (1973) Metabolism of aromatic acids by Polyporus hispidus. Phytochemistry 12: 583–587CrossRefGoogle Scholar
  35. Rider BF, Mellon MG (1946) Colorimetric determination of nitrites. Ind Eng Chem 18: 96–98Google Scholar
  36. Schweiger G, Dutscho R, Buckel W (1987) Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein. Eur J Biochem 169: 441–448CrossRefGoogle Scholar
  37. Sembiring T, Winter J (1989) Anaerobic degradation of phenylacetic acid by mixed and pure cultures. Appl Microbiol Biotechnol 31: 84–88Google Scholar
  38. Seyfried B, Tschech A, Fuchs G (1991) Anaerobic degradation of phenylacetate and 4-hydroxyphenylacetate by denitrifying bacteria. Arch Microbiol 155: 249–255CrossRefGoogle Scholar
  39. Sigoillot JC, Nguyen MH (1992) Complete oxidation of linear alkylbenzene sulfonate by bacterial communities selected from coastal seawater. Appl Environ Microbiol 58: 1308–1312PubMedPubMedCentralGoogle Scholar
  40. Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41: 100–180PubMedPubMedCentralGoogle Scholar
  41. Tschech A, Fuchs G (1987) Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads. Arch Microbiol 148: 213–217CrossRefGoogle Scholar
  42. Ward LA, Johnson KA, Robinson JM, Yokoyama MT (1987) Isolation from swine feces of a bacterium which decarboxylates p-hydroxyphenylacetic acid to 4-methylphenol (p-cresol). Appl Environ Microbiol 53: 189–192PubMedPubMedCentralGoogle Scholar
  43. Widdel F, Pfennig N (1984) Dissimilatory sulfate- or sulfurreducing bacteria. In: Krieg NR, Holt IG (eds) Bergey's manual of systematic bacteriology, vol, 9th edn. Williams & Wilkins, Baltimore London, pp 663–679Google Scholar
  44. Widdel F, Kohring GW, Mayer F (1983) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. III. Characterization of the filamentous gliding Desulfonema limicola gen. nov. sp. nov., and Desulfonema magnum sp. nov. Arch Microbiol 134: 286–294CrossRefGoogle Scholar
  45. Yokoyama MT, Carlson JR (1981) Production of skatole and para-cresol by a rumen Lactobacillus sp. Appl Environ Microbiol 41: 71–76PubMedPubMedCentralGoogle Scholar
  46. Ziegler K, Braun K, Böckler A, Fuchs G (1987) Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain. Arch Microbiol 149: 62–69CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Magdy El-Said Mohamed
    • 1
  • Birgit Seyfried
    • 1
  • Andreas Tschech
    • 1
  • Georg Fuchs
    • 1
  1. 1.Angewandte MikrobiologieUniversität UlmUlmGermany

Personalised recommendations