Desulfatiglans aniliniInitiates Degradation of Aniline With the Production of Phenylphosphoamidate and 4-Aminobenzoate as Intermediates Through Synthases and Carboxylases From Different Gene Clusters

The anaerobic degradation of aniline was studied in the sulfate-reducing bacteriumDesulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes ofD. anilinigrown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grownD. anilinicells but not in phenol- or benzoate-grown cells were located in the putative gene clustersani(aniline degradation),hcr(4-hydroxybenzoyl-CoA reductase) andphe(phenol degradation). Of these putative gene clusters, only thephegene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced inE. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene clusteraniand the other from gene clusterphe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn2+, K+, and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacteriumD. anilinionly by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, thatD. anilinicarboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.