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 formed during initial steps in the degradation of nitrobenzene to a novel ring-fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechanism 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)-chelating chromatography was used to partially purify the enzyme from an Escherichia 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 in 25 mM Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was carried out in 18O-labeled water, the product did not contain 18O, as determined by GC-MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate.
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Air Force Research Laboratory, Tyndall Air Force Base, FL 32403, USA.
Nadeau
L J
LJ
Spain
J C
JC
D000627:Aminophenols / Q000378:metabolism; D002384:Catalysis; D002827:Chorismic Acid / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006898:Hydroxylamines / Q000378:metabolism*; D006900:Hydroxylation; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D007536:Isomerism; D008970:Molecular Weight; D009584:Nitrogen / Q000378:metabolism; D017830:Octoxynol; D010103:Oxygen Isotopes; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D014867:Water / Q000378:metabolism
D000627:Aminophenols; D006898:Hydroxylamines; D010103:Oxygen Isotopes; D011993:Recombinant Fusion Proteins; D014867:Water; C027667:2-aminophenol; C012484:phenylhydroxylamine; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol; D019751:Intramolecular Transferases; D002827:Chorismic Acid; D009584:Nitrogen; C026729:4-aminophenol