Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/20/2003
Publication Date: 11/20/2003
Citation: He, Z., Nadeau, L.J., Spain, J.C. 2003. An intramoleuclar transfer of hydroxyl groups involves in enzymatic conversion of hydroxylamino aromatic compounds either to aminophenol or to dihydroxyl benzene. Applied and Environmental Microbiology.69: 2786-2793 Interpretive Summary: Nitroaromatic compounds are used for pesticides, synthetic intermediates, and explosives. On the other hand, some of these compounds are pollutants to soil and ground water. Degradation of those compounds by microorganisms is one of the strategies to clean up relevant contaminated sites. A biodegradation process is involved in a series of enzymatic reactions. Investigation of each step will enable us to understand the details of a biodegradation process. This information will be useful in the development of an effective biodegradation strategy. In this study, we examined the step of the conversion of hydroxylamino aromatic intermediates in water containing isotopic oxygen-18. Our results indicate that no water involves in this step. This finding overturns the previous accepted assumption that water takes more active part in this step. This finding is critical to set up a correct reaction mechanism in biodegradation of relevant nitroaromatic compounds.
Technical Abstract: Hydroxyl amino aromatic compounds can be enzymatically converted to either aminophenol or dihydroxyl benzene. The common characteristic of these reactions is transferring a hydroxyl group from the nitrogen atom to a carbon atom on the benzene ring. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular) or the solvent water (intermolecular). We previously reported that the conversion of hydroxylaminobenzene to 2-aminophenol proceeds by the intramolecular hydroxyl transfer mechanism. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol or 4-hydroxylaminobenzoate to 3,4-dihydroxybenzoate were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. We designed 18O -labeling experiments to reveal the hydroxyl transfer mechanisms of the two types of reactions. Contrast to the ehypothesis, our GC-MS data did not indicate any 18O labeled products in th conversion of hydroxylaminobenzene to 2- and 4-aminophenpols in H218O catalyzed by mutase from Ralstonia eutropha JMP134. In the case of conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product 3,4-dihydroxylbenzoate was 18O labeled, suggesting that another hydroxyl group in the product must have come from the substrate. Therefore, all the three types of enzymatic conversion of hydroxyl amino aromatic compounds, hydroxylaminobenzene to 2-aminophenol only, or to both 2- and 4-aminophenol, and hydroxylaminobenzoate to 3,4-dihydroxybenzoate, proceed via intramolecular hydroxyl transfer. The results set the foundation for establishment of the catalytic mechanisms for the reactions.