Cloning and Partial Characterization of an Aniline Metabolic Pathway (Preprint).

摘要

Previous studies on aniline metabolism determined that the initial enzymatic step removes the amino group and results in formation of catechol. Subsequent cleavage of the ring can occur either via the ortho or meta pathway. We report here the characterization of a Pseudomonas sp.; CIT1, that has the ability to grow on aniline, 3-methylaniline, or 4-methylaniline as the sole carbon and energy source, and cloning of the genes that govern the conversion of aniline to organic acids. The pathway resides on a 20.66 kb BamH1 fragment, and is induced by a broad range of substituted anilines, with para substituted anilines acting as the strongest inducers. The substrate range of the pathway enzymes is also broad, and includes chloro, hydroxy, and methyl substitutions, with preference to additions in the meta and para positions. Metabolism of aniline in CIT1 is initiated by aniline, 1,2 dioxygenase, and results in a stoichiometric release of ammonia and putative formation of catechol. This initial dioxygenation was indicated by quantitive respirometry; aniline required 3.14 moles O2/mol aniline while catechol requires 2.12 moles O2/mole catechol. The one mole difference is consistent with a dioxygenation reaction. The ring is cleaved by catechol 2,3 dioxygenase to form a yellow compound with an absorbance maximum at 375 nm, which is consistent with 2-hydroxymuconic semialdehyde. This is further metabolized by an NAD-dependent dehydrogenase and an NAD-independent hydrolase. Aniline metabolism in E.coli, expressing the cloned pathway was confirmed using HPLC.