Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45

Abstract

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 18 O-labeled water, the product did not contain 18 O, 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.Keywords: Bamberger rearrangement; hydroxy transfer; hydroxylaminobenzene; hydroxymutase; SDS stability.The most common pathways for biodegradation of nitroaromatic compounds involve removal of the nitrogen before ring cleavage by release of either nitrite (oxidative pathway, e.g. [1,2]) or ammonia (reductive pathway, e.g. [3±5]). In the latter pathway, ammonia is released from a hydroxylamino intermediate to form the corresponding catechol. During degradation of nitrobenzene [6], chloronitrobenzene [7,8], 3-nitrophenol [9], and 4-nitrotoluene [10] by some bacteria, the hydroxylamino intermediates undergo an enzymecatalyzed rearrangement to the corresponding ortho-aminophenols (Fig. 1). The ortho-aminophenolic compounds are further degraded via ring cleavage pathways to central metabolites [11±14].In organic chemistry, the conversion of hydroxylamino aromatic compounds into aminophenols is called the Bamberger rearrangement [15]. In its simplest form, the reaction is carried out in aqueous sulfuric acid and yields 4-aminophenol as the major product [15]. The interest in the Bamberger rearrangement is due to the fact that such reactions are observed not only in the reduction of nitroaromatic compounds, but also in the oxidation of aromatic amines and a number of biologically important processes [8,16±19].Although the biological conversion of hydroxylamino compounds has also been referred to as Bamberger or Bambergerlike rearrangement, ortho-aminophenolic compounds are the predominant products, in contrast with the non enzymatic acidcatalyzed reactions.Recently, Schenzle et al. [20] reported purification and some properties of 3-hydroxylaminophenol mutase, wh...

The Enzymology of Organic Transformations: A Survey of Name Reactions in Biological Systems

“…This result suggests that the hydroxyl group in the product is not derived from solvent but rather from the intramolecular transfer of the hydroxyl group on the hydroxylamino moiety of the substrate (Figure 64b). [242] In contrast to the Bamberger-like reactions described above, in which the ortho position of the hydroxylamino substrate is hydroxylated, 3-hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a bona-fide Bamberger rearrangement. [243] The reaction catalyzed by the purified enzyme produced both the ortho (Bamberger-like) and para (Bamberger) hydroxylaminobenzene.…”

Biocatalysis: A Status Report

“…The foremost strength of biocatalysts is their often superb specificity: enzymes often far surpass other catalysts with respect to chemical specificity; regioselectivity, such as toluene monooxygenase (20), hydroxyaminobenzene (HAB) mutase (21), or cytochrome P450 BM-3 (22); or enantioselectivity, for which there are legions of examples, such as N-acyl amino acid hydrolase (acylase) (23), lipase (24), epoxide hydrolase (25), amino acid dehydrogenase (26), or amine dehydrogenase (14,27).…”

Die Enzymologie organischer Umwandlungen: Namensreaktionen in biologischen Systemen

“…Wird die Mutasereaktion in Pufferlösung mit 18 O‐markiertem Wasser durchgeführt, ist das erhaltene Produkt 2‐Aminophenol ( 241 ) nicht markiert. Daraus lässt sich schließen, dass die Hydroxygruppe im Produkt nicht aus dem Lösungsmittel stammt, sondern vielmehr intramolekular von der Hydroxylaminoeinheit des Substrats übertragen wird (Abbildung b) . Im Unterschied zu den oben beschriebenen Bamberger‐ähnlichen Reaktionen, bei denen die ortho ‐Position des Hydroxylaminosubstrats hydroxyliert wird, katalysiert 3‐Hydroxylaminophenol‐Mutase aus Ralstonia eutropha JMP134 eine echte Bamberger‐Umlagerung .…”

“…Daraus lässt sich schließen, dass die Hydroxygruppe im Produkt nicht aus dem Lçsungsmittel stammt, sondern vielmehr intramolekular von der Hydroxylaminoeinheit des Substrats übertragen wird (Abbildung 64 b). [242] Im Unterschied zu den oben beschriebenen Bamberger-ähnlichen Reaktionen, bei denen die ortho-Position des Hydroxylaminosubstrats hydroxyliert wird, katalysiert 3-Hydroxylaminophenol-Mutase aus Ralstonia eutropha JMP134 eine echte Bamberger-Umlagerung. [241] Die durch das gereinigte Enzym katalysierte Reaktion liefert das (Bamberger-ähnliche) ortho-u nd das para-Hydroxyaminobenzol (Bamberger-Produkt).…”