Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function
- 245 Downloads
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.
KeywordsBenzoate 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).
- 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
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith J, Struhl K (1987) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
- Gross GG, Zenk MH (1966) Darstellung und Eigenschaften von Coenzyme A-Thioestern substituierter Zimtseaure. Z Naturforsch B 21:683–690Google Scholar