Two similar gene clusters coding for enzymes of a new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium Azoarcus evansii.
In the beta-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 beta-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 beta-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.
Schühle
K
K
Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
Jahn
M
M
Ghisla
S
S
Fuchs
G
G
D000332:Aerobiosis; D000595:Amino Acid Sequence; D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D009876:Operon; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D006899:Mixed Function Oxygenases; C062023:2-aminobenzoyl-CoA monooxygenase-reductase; C070685:2-aminobenzoate coenzyme A ligase; D003066:Coenzyme A Ligases