A 2-COMPONENT MONOOXYGENASE CATALYZES BOTH THE HYDROXYLATION OF P-NITROPHENOL AND THE OXIDATIVE RELEASE OF NITRITE FROM 4-NITROCATECHOL IN BACILLUS-SPHAERICUS-JS905

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 THE are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol, Here we describe the com plete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil i n India. Washed cells of PNP-grown JS905 released nitrite in stoichiom etric 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 subs equently oxidized to THE with the concomitant removal of the nitro gro up as nitrite, The enzyme that catalyzed the two sequential monooxygen ations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography, B oth components were required for NADH-dependent oxidative release of n itrite from PNP or 4-nitrocatechol. One of the components was identifi ed as a reductase based on its ability to catalyze the NAD(P)H-depende nt reduction of 2,6 dichlorophenolindophenol and nitroblue tetrazolium . Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THE are catalyzed by a single two-compo nent enzyme system comprising a flavoprotein reductase and an oxygenas e.