Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function.
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.
Mikrobiologie, Fakultät für Biologie, Universität Freiburg, Freiburg, Germany. ismail@microbio.umass.edu
Wael
W
Ismail
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000332:Aerobiosis; D000693:Anaerobiosis; D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D023061:Gene Order; D005798:Genes, Bacterial; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008970:Molecular Weight; D005810:Multigene Family; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D001565:Benzoates; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C058482:4-hydroxybenzoyl-coenzyme A; D000105:Acetyl Coenzyme A; C037816:phenylacetyl-coenzyme A; C092167:4-hydroxybenzoyl-CoA hydrolase; D013869:Thiolester Hydrolases