Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45 - A highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups

Hydroxylaminobenzene mutase is the enzyme that converts intermediates forme d during initial steps in the degradation of nitrobenzene to a novel ring-f ission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase cata lyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechani sm of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap-SP, Hitrap-Q and Cu(II)-chelat ing chromatography was used to partially purify the enzyme from an Escheric hia coli clone. The partially purified enzyme was eluted in the void volume of a Superose-12 gel-filtration column even in the presence of 0.05% SDS i n 25 mh Tris/HCl buffer, which indicated that it was highly associated. Whe n the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was car ried out in O-18-labeled water, the product did not contain O-18, as determ ined by GC-MS. The results indicate that the reaction proceeded by intramol ecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular tra nsfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate.