Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: Studies on the non-oxygenolytic ring cleavage enzyme

Journal
Molecular microbiology  
Volume
56
Issue
6
Start Page
1586
End Page
1600
Citation
Molecular Microbiology 56 (6): 1586-1600 (2005-06-01)
Publisher DOI
10.1111/j.1365-2958.2005.04637.x
Scopus ID
2-s2.0-20344385268
PubMed ID
15916608
Publisher
Wiley-Blackwell
A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to benzoyl-CoA. The further down-stream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl-CoA is oxidized to 2,3-dihydro-2,3-dihydroxybenzoyl-CoA (benzoyl-CoA dihydrodiol) by benzoyl-CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxCmalBoxC) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3-dihydro-2,3-dihydroxybenzoyl-CoA + H2O →3,4-dehydroadipyl- CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring-13C6]benzoyl-CoA, benzoyl-CoA oxygenase/reductase, BoxCmal protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6-13C5]3,4- dehydroadipyl-CoA semialdehyde; the missing one-carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6-13C5]3,4-dehydroadipyl-CoA semi-aldehyde and [2,3,4,5,6-13C5]4,5-dehydroadipyl-CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed benzoyl-CoA- dihydrodiol lyase. It contains domains characteristic for enoyl-CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring-cleavage reaction does not require molecular oxygen.
DDC Class
570: Biowissenschaften, Biologie