A two-component monooxygenase catalyzes both the hydroxylation of p-nirophenol and the oxidative release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905
- Air Force Research Lab., Tyndall AFB, FL (United States)
Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here the authors describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Their results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.
- OSTI ID:
- 642322
- Journal Information:
- Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 7 Vol. 64; ISSN AEMIDF; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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