Benzoyl-coenzyme A thioesterase of Azoarcus evansii : properties and function
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ORIGINAL PAPER
Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function Wael Ismail
Received: 26 February 2008 / Revised: 23 April 2008 / Accepted: 19 May 2008 / Published online: 10 June 2008 © Springer-Verlag 2008
Abstract 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-dihydroxybenzoylCoA, 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.
Communicated by Ercko Stackebrandt. W. Ismail Mikrobiologie, Fakultät für Biologie, Universität Freiburg, Freiburg, Germany W. Ismail (&) Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA e-mail: [email protected]
Keywords Benzoate metabolism · Thioesterase · Benzoyl-coenzyme A
Introduction The aerobic metabolism of aromatic compounds follows well-established pathways (for a recent review see Harwood and Parales 1996). Initially, mono- or dioxygenases introduce hydroxyl groups into the ring of the various aromatic substrates, thus producing few key intermediates such as catechol, protocatechuate, gentisate, or homogentisate. These are substrates for subsequent ring-cleaving dioxygenases (reviewed by Butler and Mason 1997). The aliphatic products of ring Wssion are further transformed to central metabolites such as acetyl-coenzyme A (CoA) and succinyl-CoA. However, the aerobic metabolism of benzoate by some bacteria does not follow this catabolic strategy. Benzoate metabolism by Azoarcus evansii and other bacteria from diverse groups is mediated by an unorthodox pathway which combines features from both the classical aerobic (described above) as well as the anaerobic pathways for metabolism of aromatic compounds (Fig. 1a) (Zaar et al. 2001). Besides an oxygen dependent hydroxylation step similar to the aerobic strategy, the novel route has the following features which are common to anaerobic metabolism of mono-aromatics (Boll et al. 2002); (1)
