Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium Azoarcus evansii

Abstract

In the ␤-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a ␤-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by ␤-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.2-Aminobenzoic acid (anthranilic acid) plays an important role in the synthesis and degradation of many N-heterocyclic compounds such as tryptophan, indole, indoleacetic acid, and derived compounds. As a consequence of its wide occurrence, 2-aminobenzoate is a common substrate for many microorganisms that are able to cleave aromatic rings.There are three established aerobic pathways by which 2-aminobenzoate is converted to either catechol or gentisate (reviewed in reference 26). These compounds are central intermediates of aerobic aromatic metabolism which are further oxidized and cleaved, via either ortho-or meta-cleavage pathways. Notably, the aromatic ring is cleaved oxygenolytically, and coenzyme A (CoA) thioesters of organic acids come into play only after ring cleavage, e.g., as 3-oxo-adipyl-CoA and succinyl-CoA. Such rules also apply to the aerobic metabolism of other aromatic substrates. These pathways have been discussed and reviewed elsewhere (14).

ThebzdGene Cluster, Coding for Anaerobic Benzoate Catabolism, inAzoarcussp. Strain CIB

“…As shown for other benzoate degraders, the anaerobic catabolism of benzoate begins with the activation of the aromatic substrate to benzoyl-CoA by the action of a benzoate-CoA ligase (AMP forming) (1,27,55) (Fig. 1).…”

Benzoate-Coenzyme A Ligase from Thauera aromatica : an Enzyme Acting in Anaerobic and Aerobic Pathways

“…Their functions are completely unknown. Genes similar to ORF 3, a member of the MarR family, have been found in gene clusters responsible for anaerobic 4-hydroxybenzoate metabolism in T. aromatica (13) and for aerobic 2-aminobenzoate metabolism in A. evansii (34). In R. palustris, two proteins, AadR and BadR, regulate expression of benzoylCoA reductase and probably other components of the general benzoyl-CoA pathway in response to oxygen and benzoate, respectively (15).…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…In A. evansii, the aerobic degradation of 2-aminobenzoate involves the initial activation to 2-aminobenzoyl-CoA carried out by specific aerobic CoA ligases. 2-Aminobenzoyl-CoA is subsequently hydroxylated and reduced to a nonaromatic product by the flavoenzyme 2-aminobenzoyl-CoA monooxygenase/reductase (331). Whereas two gene clusters responsible for the hybrid pathway for the aerobic degradation of 2-aminobenzoate have been characterized for A. evansii (331), most of the genes involved in the anaerobic 2-aminobenzoate degradation pathway still remain unknown.…”

“…bacteria have been described, and some of these gene products have been biochemically characterized (100,224,330). The substrate specificity of benzoate-CoA ligases can differ depending on the host strain, but it is common that these enzymes can recognize and activate halobenzoates (5,123,181,224,330), and in some cases, they are also responsible for the initial step in the anaerobic catabolism of benzoate derivatives such as 2-aminobenzoate (331). On the other hand, other aromatic/alicyclic acid-CoA ligases can also recognize benzoate as a substrate, and for instance, R. palustris cells synthesize at least three different enzymes, i.e., benzoate-CoA ligase (BadA), 4-hydroxybenzoate (4-HBA)-CoA ligase (HbaA), and cyclohexanecarboxylate-CoA ligase (AliA), that can catalyze the activation of benzoate to benzoyl-CoA during anaerobic growth with benzoate (100).…”