A 2-COMPONENT MONOOXYGENASE CATALYZES BOTH THE HYDROXYLATION OF P-NITROPHENOL AND THE OXIDATIVE RELEASE OF NITRITE FROM 4-NITROCATECHOL IN BACILLUS-SPHAERICUS-JS905
V. Kadiyala et Jc. Spain, A 2-COMPONENT MONOOXYGENASE CATALYZES BOTH THE HYDROXYLATION OF P-NITROPHENOL AND THE OXIDATIVE RELEASE OF NITRITE FROM 4-NITROCATECHOL IN BACILLUS-SPHAERICUS-JS905, Applied and environmental microbiology, 64(7), 1998, pp. 2479-2484
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.