Archives of Microbiology

, Volume 190, Issue 4, pp 451–460 | Cite as

Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function

Original Paper


The aerobic benzoate metabolism in Azoarcus evansii follows an unusual route. The intermediates of the pathway are processed as coenzyme A (CoA) thioesters and the cleavage of the aromatic ring is non-oxygenolytic. The enzymes of this pathway are encoded by the box gene cluster which harbors a gene, orf1, coding for a putative thioesterase. Benzoyl-CoA thioesterase activity (20 nmol min−1 mg−1 protein) was present in cells grown aerobically on benzoate, but was lacking in cells grown on other aromatic or aliphatic substrates under oxic or anoxic conditions. The gene was cloned and overexpressed in Escherichia coli to produce a C-terminal His-tag fusion protein. The recombinant enzyme was a homotetramer of 16 kDa subunits. It catalyzed not only the hydrolysis of benzoyl-CoA, but also of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA, the second intermediate in the pathway. The enzyme exhibited higher activity with mono-substituted derivatives of benzoyl-CoA, showing highest activity with 4-hydroxybenzoyl-CoA. Di-substituted derivatives of benzoyl-CoA, phenylacetyl-CoA, and aliphatic CoA thioesters were not hydrolyzed but some acted as inhibitors. The thioesterase appears to protect the cell from CoA pool depletion. It may constitute the prototype of a new subfamily within the hotdog fold enzyme superfamily.


Benzoate metabolism Thioesterase Benzoyl-coenzyme A 



I am very grateful to Georg Fuchs for his support, fruitful discussions and critical reading of the manuscript. I am indebted to Matthias Boll for kindly offering aromatic CoA thioesters. I gratefully acknowledge the financial support of this work by the Deutsche Forschungsgemeinschaft (DFG).


  1. Anders JH, Kätze A, Kämpfer P, Ludwig W, Fuchs G (1995) Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K172 and KB740 and their description as new members of genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of proteobacteria. Int J Syst Bacteriol 45:327–333PubMedCrossRefGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith J, Struhl K (1987) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  3. Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds. 4-Hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species. Eur J Biochem 213:555–561PubMedCrossRefGoogle Scholar
  4. Boll M, Fuchs G, Heider J (2002) Anaerobic oxidation of aromatic compounds and hydrocarbons. Curr Opin Chem Biol 6:604–611PubMedCrossRefGoogle Scholar
  5. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Butler CS, Mason JR (1997) Structure-function analysis of the bacterial aromatic ring-hydroxylating dioxygenases. Adv Microb Physiol 38:47–84PubMedCrossRefGoogle Scholar
  7. Cheng J, Randall M, Sweredoski PB (2005) A protein structure and structural feature prediction server. Nucleic Acids Res 33:72–76CrossRefGoogle Scholar
  8. Dillon CS, Betaman A (2004) The hotdog fold: wrapping up a superfamily of thioesterases and dehydratases. BMC Bioinformatics 5:109–122PubMedCrossRefGoogle Scholar
  9. Fairley DJ, Wang G, Rensing C, Pepper IL, Larkin MJ (2006) Expression of gentisate 1, 2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera. Appl Microbiol Biotechnol 73:691–695PubMedCrossRefGoogle Scholar
  10. Gescher J, Zaar A, Mohamed M, Schägger H, Fuchs G (2002) Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii. J Bacteriol 184:6301–6315PubMedCrossRefGoogle Scholar
  11. Gescher J, Eisenreich W, Wörth J, Bacher A, Fuchs G (2005) Aerobic benzoyl-CoA in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme. Mol Microbiol 56:1586–1600PubMedCrossRefGoogle Scholar
  12. Gescher J, Ismail W, Oelgeschläger E, Eisenreich W, Wörth J, Fuchs G (2006) Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii. Conversion of ring cleavage product by 3, 4-dehydroadipyl-CoA semialdehyde dehydrogenase. J Bacteriol 188:2919–2927PubMedCrossRefGoogle Scholar
  13. Gross GG, Zenk MH (1966) Darstellung und Eigenschaften von Coenzyme A-Thioestern substituierter Zimtseaure. Z Naturforsch B 21:683–690Google Scholar
  14. Harwood CS, Parales RE (1996) The β-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50:553–590PubMedCrossRefGoogle Scholar
  15. Ismail W, Mohamed ME, Wanner BL, Datsenko KA, Eisenreich W, Rohdich F, Bacher A, Fuchs G (2003) Functional genomics by NMR spectroscopy: phenylacetate catabolism in Escherichia coli. Eur J Biochem 270:3047–3054PubMedCrossRefGoogle Scholar
  16. Jackowski S, Rock CO (1986) Consequences of reduced intracellular coenzyme A content in Escherichia coli. J Bacteriol 166:866–871PubMedGoogle Scholar
  17. Kunishima N, Asada Y, Sugahara M, Ishijima J, Nodake Y, Sugahara M, Miyano M, Kuramitsu S, Yokoyama S, Sugahara M (2005) A novel induced-fit reaction mechanism of asymmetric hotdog thioesterase PaaI. J Mol Biol 352:212–228PubMedCrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Lochmeyer C, Koch J, Fuchs G (1992) Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium. J Bacteriol 174:3621–3628PubMedGoogle Scholar
  20. Lopez Barragan MJ, Carmona M, Zamarro MT, Thiele B, Boll M, Fuchs G, Garcia JL, Diaz E (2004) The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. strain CIB. J Bacteriol 186:5762–5774PubMedCrossRefGoogle Scholar
  21. Mohamed ME, Seyfried B, Tschech A, Fuchs G (1993) Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in the denitrifying Pseudomonas sp. Evidence for an α-oxidation mechanism. Arch Microbiol 159:563–573CrossRefGoogle Scholar
  22. Mohamed ME, Ismail W, Heider J, Fuchs G (2002) Aerobic metabolism of phenylacetic acids in Azoarcus evansii. Arch Microbiol 178:180–192CrossRefGoogle Scholar
  23. Parke D, Ornston LN (2004) Toxicity caused by hydroxycinnamoyl-coenzyme A thioester accumulation in mutants of Acinetobacter sp. strain ADP1. Appl Environ Microbiol 70:2974–2983PubMedCrossRefGoogle Scholar
  24. Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27–36PubMedCrossRefGoogle Scholar
  25. Ren Y, Aguirre J, Ntamack AG, Chu C, Schulz H (2004) An alternative pathway of oleate β-oxidation in Escherichia coli involving the hydrolysis of a dead end intermediate by a thioesterase. J Biol Chem 279:11042–11050PubMedCrossRefGoogle Scholar
  26. Schneider S, Mohamed ME, Fuchs G (1997) Anaerobic metabolism of l-phenylalanine via benzoyl-coenzyme A in the denitrifying bacterium Thauera aromatica. Arch Microbiol 168:310–320PubMedCrossRefGoogle Scholar
  27. Schuehle K, Jahn M, Ghisla S, Fuchs G (2001) Two similar gene clusters coding for enzymes of a new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium Azoarcus evansii. J Bacteriol 183:5268–5278CrossRefGoogle Scholar
  28. Song F, Zhuang Z, Finci L, Dunaway-Mariano D, Kniewel R, Buglino JA, Solorzano V, Wu J, Lima CD (2006) Structure, function, and mechanism of the phenylacetate pathway hotdog-fold thioesterase PaaI. J Biol Chem 281:11028–11038PubMedCrossRefGoogle Scholar
  29. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  30. Zaar A, Eisenreich W, Bacher A, Fuchs G (2001) A novel pathway of aerobic benzoate catabolism in the bacteria Azoarcus evansii and Bacillus stearothermophilus. J Biol Chem 276:24997–25004PubMedCrossRefGoogle Scholar
  31. Zaar A, Gescher J, Eisenreich W, Bacher A, Fuchs G (2004) New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii. Mol Microbiol 54:223–238PubMedCrossRefGoogle Scholar
  32. Zhuang Z, Song F, Zhang W, Taylor K, Archambault A, Dunaway-Mariano D (2002) Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS-3 4-hydroxybenzoyl-CoA thioesterase active site. Biochemistry 41:11152–11160PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Mikrobiologie, Fakultät für BiologieUniversität FreiburgFreiburgGermany
  2. 2.Department of MicrobiologyUniversity of MassachusettsAmherstUSA

Personalised recommendations