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Item type:Publication, Access status: without files , Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: Conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase(American Society for Microbiology, 2006-04-15); ; ; ; Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-deliydroadipyl-CoA semialdehyde was studied in the β-proteobacterium Azoarcws evansii. Cell extracts contained a benzoate-induced, NADP+-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3- dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.Publicationtype: Journal ArticleScopus© Citations 33 18 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: Studies on the non-oxygenolytic ring cleavage enzyme(Wiley-Blackwell, 2005-04-29); ; ; ; 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.Publicationtype: Journal ArticleScopus© Citations 43 11 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii(Wiley-Blackwell, 2004-08-11); ; ; ; A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways. In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl-CoA, which is subsequently attacked by an oxygenase, followed by a non-oxygenolytic fission of the ring. We provide evidence for this hypothesis and show that benzoyl-CoA conversion in the bacterium Azoarcus evansii requires NADPH, O 2 and two protein components, BoxA and BoxB. BoxA is a homodimeric 46 kDa iron-sulphur-flavoprotein, which acts as reductase. In the absence of BoxB, BoxA catalyses the benzoyl-CoA stimulated artificial transfer of electrons from NADPH to O2 via free FADH2 to produce H2O 2. Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron-protein that acts as benzoyl-CoA oxygenase. The product of benzoyl-CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA. This suggests that BoxBA act as a benzoyl-CoA dioxygenase/reductase. Unexpectedly, benzoyl-CoA transformation by BoxBA was greatly stimulated when another enoyl-CoA hydratase/isomerase-like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl-CoA. The benzoyl-CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.Publicationtype: Journal ArticleScopus© Citations 40 15 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Benzoate-coenzyme a ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways(American Society for Microbiology, 2003-08-15); ; ; ; In the denitrifying member of the β-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on Vmax/Km, the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.Publicationtype: Journal ArticleScopus© Citations 70 12 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii(American Society for Microbiology, 2002-11-15); ; ; ; A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the β-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for β-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A β-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.Publicationtype: Journal ArticleScopus© Citations 93 18