Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups.

Nitroaromatic compounds can be degraded either oxidatively or reductively by microorganisms (25). Hydroxylamino intermediates are produced from nitroaromatic compounds in reactions catalyzed by nitroreductases in a number of bacteria (25). The hydroxylamino intermediates are then degraded via one of three different routes. In compounds with more than one nitro group, the intermediate is often reduced to the amine, as in the transformation of 2,4,6-trinitrotoluene (16, 28). In a second strategy, used during degradation of 4-nitrobenzoate (4-NBA) by a variety of isolates (6, 9, 27, 29, 35), the hydroxylamino compound is converted to protocatechuate in a reaction catalyzed by a lyase enzyme. A third strategy used by bacteria is the rearrangement of the hydroxylamino intermediates to the corresponding aminophenols (APs) as in the degradation of nitrobenzene (24, 26), 3-nitrophenol (30), chloronitrobenzene (17), 4-nitrotoluene (33), 2,4,6-trinitrotoluene (5, 14), and 2-NBA (10). Most of the APs can be further metabolized by an extradiol-like ring cleavage pathway, as observed in the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 (19) and Pseudomonas putida HS12 (26), the degradation of 4-nitrotoluene by Mycobacterium sp. strain HL 4-NT-1 (13, 33), and by 3-hydroxyanthranilate 3,4-dioxygenase in Pseudomonas fluorescens strain KU-7 (T. Muraki, T. M., Y. Hasegawa, H. Iwaki, and P. C. K. Lau, Abstr. Am. Soc. Microbiol. Conf. Biodegradation Biotransformation Biocatalysis, abstr. 62, p. 42, 2001). There are various possibilities for the transfer of the hydroxyl groups during conversion of hydroxylamino aromatic intermediates (Table ​(Table1).1). The origin of the hydroxyl group of the products could be the substrate (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The acid-catalyzed Bamberger rearrangement of hydroxylaminobenzene (HAB) to 4-AP proceeds via the intermolecular transfer mechanism (18) (Table ​(Table1).1). Recombinant Escherichia coli strains carrying the HAB mutase gene (habB) cloned from P. pseudoalcaligenes JS45 catalyze an intramolecular transfer of the hydroxyl group of HAB to form 2-AP (11). HAB mutase is potentially useful as a biocatalyst for the synthesis of o-APs (23). A second mutase gene, habA, was cloned from strain JS45, and subsequent genetic and biochemical experiments revealed that only habA is expressed in the wild type, but it is not known whether the enzyme catalyzes a similar intramolecular reaction (4). The mutase from Ralstonia eutropha JMP134 catalyzes the transformation of HAB to a mixture of 2- and 4-AP (31). It has been proposed that the reaction mechanism from the mutase from strain JMP134 is analogous to the acid-catalyzed Bamberger rearrangement because the enzyme requires no oxygen or added cofactors for its reaction (31). Considering the fact that the products in the reactions catalyzed by the JMP134 mutase are not limited to the one produced by either the Bamberger rearrangement or by the JS45 mutase (Table ​(Table1),1), the hypothesis must be tested experimentally. TABLE 1. Mechanisms of the conversion of hydroxylamino aromatic compounds 4-Hydroxylaminobenzoate (4-HABA) lyase catalyzes the transformation of 4-HABA to protocatechuate in Pseudomonas strain 4NT and several other isolates (6, 9, 29, 35). In the protocatechuate product, one of the hydroxyl groups must originate from water, but the other could be either from water or from the substrate, 4-HABA. Meulenberg and de Bont (22) proposed that the hydroxyl group of 4-HABA is first released and then two hydroxyl groups from water are added to the benzene ring with the release of ammonia to form the product, protocatechuate (Table ​(Table11). We sought to determine here whether the intramolecular mechanism observed in the conversion of HAB by HabB mutase in strain JS45 enzyme also applies to the two types of enzyme-catalyzed reactions that were previously thought to involve intermolecular transfer of hydroxyl groups. We have determined unequivocally the origin of the hydroxyl groups in the reaction products to provide insight into the reaction mechanism.