Previous Article | Next Article 
Applied and Environmental Microbiology, April 2000, p. 1474-1478, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
2,4,6-Trinitrotoluene Reduction by Carbon Monoxide
Dehydrogenase from Clostridium thermoaceticum
Shouqin
Huang,1
Paul A.
Lindahl,2
Chuanyue
Wang,3
George N.
Bennett,1,*
Frederick B.
Rudolph,1 and
Joseph
B.
Hughes3
Department of Biochemistry and Cell Biology
and the Institute of Biosciences and
Bioengineering1 and Department of
Environmental Science and Engineering,3 Rice
University, Houston, Texas 77005-1892, and The Departments of
Chemistry and of Biochemistry and Biophysics, Texas A&M University,
College Station, Texas 778432
Received 24 September 1999/Accepted 19 January 2000
 |
ABSTRACT |
Purified CO dehydrogenase (CODH) from Clostridium
thermoaceticum catalyzed the transformation of
2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of
TNT transformation were separated and appear to be identical to the
compounds formed by C. acetobutylicum, namely,
2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT),
4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT),
2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger
rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the
presence of saturating CO, CODH catalyzed the conversion of TNT to two
monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as
the dominant isomer. These derivatives were then converted to 24DHANT,
which slowly converted to the Bamberger rearrangement product. Apparent
Km and kcat values of
TNT reduction were 165 ± 43 µM for TNT and 400 ± 94 s
1, respectively. Cyanide, an inhibitor for the
CO/CO2 oxidation/reduction activity of CODH, inhibited the
TNT degradation activity of CODH.
| |
INTRODUCTION |
2,4,6-Trinitrotoluene (TNT) is a
chemical explosive that is a common contaminant of soils and
groundwater at numerous Department of Defense facilities. For several
decades, research has been conducted to develop ecologically sound
means of remediating sites contaminated with this toxic compound.
Studies investigating the potential for bioremediation (i.e., the use
of microorganisms to metabolize hazardous wastes) have demonstrated
that many aerobic and anaerobic microorganisms are capable of
catalyzing the reduction of aryl nitro groups associated with TNT
(3, 6, 8-10, 16), forming often uncharacterized products.
Aerobic microorganisms contain nitroreductases that catalyze such
reductions. An aryl nitro reductase purified from Neurospora crassa reduces several nitrobenzenes, 3,5-dinitrobenzoic acid, and
TNT (22). In the presence of NADPH, enzymes in the crude extract of Pseudomonas sp. strain CBS3 catalyzed the
reduction of p-nitrobenzoate and TNT (16). An
NADH- or NADPH-dependent nitro reductase from Enterobacter
cloacae also catalyzes the reduction of TNT (3).
Some anaerobes reduce nitro groups to amines through a mechanism
involving nitroso and hydroxyl-amino intermediates, while others, such
as Clostridium acetobutylicum, reduce the nitro groups of
TNT to hydroxyl-amino-nitrotoluenes without further reduction to the
corresponding amines (6-8). The enzymes and/or proteins involved in these transformations have not been identified (9, 10), although ferredoxins, hydrogenases, CO dehydrogenases
(CODHs), pyruvate-ferredoxin oxidoreductases, and sulfite reductases
have all been implicated. For example, hydrogenase from
Clostridium pasteurianum (in the presence of H2)
and partially purified CODH from Clostridium thermoaceticum
(in the presence of CO), reduced 2,4-diamino-6-nitrotoluene to
2,4-diamino-6-hydroxylaminotoluene when ferredoxin was included in the
reaction mixture (10). Reduction also occurred with reduced
ferredoxin or methyl viologen in the absence of enzymes, although the
rate was slower by orders of magnitude (10). These results
suggested that hydrogenase and CODH may act to reduce nitroaromatic
compounds by reduction of ferredoxin or methyl viologen. This premise
is congruent with the ability of numerous nonspecific agents
(H2 on Pt, Zn, and sulfide) to reduce aryl nitro groups.
The direct action of these enzymes on TNT, which has multiple nitro
groups, has not been described previously.
CODHs from homoacetogens such as Clostridium thermoaceticum
also catalyze the synthesis of acetyl-coenzyme A (from CO, a methyl group, and coenzyme A [CoA]) (13). The CO/CO2
redox activity occurs at a novel Ni-X-Fe4S4
cluster known as the C cluster (1, 5), while acetyl-CoA
synthesis occurs at a different Ni-Y-Fe4S4 cluster known as the A cluster (14, 20, 21). X and Y are unidentified ligands that bridge the Ni and
Fe4S4 components of these distinct clusters.
The primary purpose of studies reported here was to determine whether
CODH directly catalyzes TNT reduction or serves only to transfer
electrons to other species that reduce TNT nonspecifically. In this
report, we show that CODH from C. thermoaceticum catalyzes the reduction of TNT in the presence of CO and that it does so in the
absence of ferredoxins or viologens. The reductase activity saturated
at increasing TNT concentrations, allowing Km
and kcat determinations, and was inhibited by
cyanide ion (a known inhibitor for the CO/CO2 redox
activity of the enzyme). These results were used to propose the
preliminary catalytic mechanism for the CODH-catalyzed reduction of TNT.
| |
MATERIALS AND METHODS |
Materials.
TNT was purchased from Chem Service (West
Chester, Pa.). [U-ring-14C]TNT was obtained from Chemsyn
Science (Lenexa, Kans.). 2-Hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and
2,4-dihydroxylamino-6-nitrotoluene were synthesized from
biotransformation of TNT using C. acetobutylicum and
characterized by mass spectroscopy, 1H nuclear magnetic
resonance, and infrared spectroscopy (6). Solvents of
high-pressure liquid chromatography (HPLC)-grade methanol and
2-propanol were obtained from Fisher Scientific. Perchloric acid was
purchased from Mallinckrodt (St. Louis, Mo.). Potassium cyanide (Baker)
was prepared in 50 mM NaOH solutions.
Purification of CODH.
CO dehydrogenase was isolated and
purified anaerobically as described earlier (11, 17). The
final purity was >90% based on the visual estimate of a sodium
dodecyl sulfate-polyacrylamide gel. Ferredoxin was cleanly separated
from CODH during the gradient DEAE step of the purification, as it
remains bound beyond the highest salt concentration used (0.4 M NaCl)
(12). Moreover, the electron paramagnetic resonances
characteristic of eight-iron ferredoxins were not observed in the
spectra of dithionite-reduced samples. All experiments involving the
enzyme were performed anaerobically either in a Vacuum/Atmospheres
Ar-Atmosphere glove box or a Forma Scientific glove box (90:10
[vol/vol] N2 and H2, respectively). After
purification, the enzyme was passed through a Sephadex G25 column
equilibrated in 20 mM Tris (pH 8.0) and 10 mM dithiothreitol.
Analysis of TNT and other nitroaromatic compounds.
TNT and
its metabolites were fractionated and quantified by using a Waters
(Milford, Mass.) HPLC apparatus with diode array UV-visible (UV-VIS)
detection and the Millenium Chromatography manager. Spectra were
continuously acquired at between 200 and 400 nm, and chromatograms were
extracted at 230 nm for quantification. Analytes were separated on a
reversed-phase Waters Nova-Pak C8 column (3.9 by 150 mm) at
room temperature with an isocratic mobile-phase mixture of 82% water
and 18% 2-propanol at 1 ml/min. This system has been well established
for separation of hydroxylamino derivatives of TNT reduction compared
to related aminated forms (6-8, 19). Peak identification
was based on the comparison of elution times and UV spectra with
authentic standards.
TNT transformation assay.
CODH (1.8 to 110 µg) was
injected into septum-sealed vials containing 2 ml of 50 mM Tris-HCl (pH
7.9) and various concentrations of TNT (41 to 210 µM) at room
temperature (~27°C) under 1 atm of CO. At increasing times after
adding CODH, 100-µl aliquots were removed, mixed with 10 µl of 70%
perchloric acid to quench the reaction, and analyzed by HPLC.
Purification of [U-ring-14C]TNT.
A solution of
[U-ring-14C]TNT in methanol (106 dpm/ml) was
purified by silica column chromatography (ethyl acetate hexane, 1:6 [vol/vol]). An aliquot containing the purified
[U-ring-14C]TNT was dried by evaporation, and the residue
was dissolved in methanol to obtain a stock solution (105
dpm/ml) of [U-ring-14C]TNT. The purity, as evaluated by
HPLC fractionation (using the HPLC method), was 98.6%. The stock (20 µl) was mixed with unlabeled TNT (10 ml of a 10-mg/ml concentration)
also in methanol to prepare secondary stock solutions.
Kinetics of TNT transformation.
The secondary stock solution
(10 µl) was used in the TNT degradation assays described above.
Certain processes occurred more rapidly than others, and so to probe
both slow and rapid processes different amounts of CODH were used. For
assays quenched at times less than 20 min (probing the rapid
processes), 1.8 µg of CODH was employed, while 110 µg was used for
assays quenched at later times (probing the slow processes). Reaction
rates were assumed to be proportional to CODH concentration; thus, the
results of the two sets of experiments were normalized to the CODH
concentrations used. The concentration profiles of HPLC-detectable
intermediates and products were monitored (between 200 and 400 nm) as a
function of reaction time. Aliquots of HPLC fractions (both peaks and
background regions) were added directly to 5 ml of scintillation
cocktail (Ready Gel; Beckman, Fullerton, Calif.), which were then
counted (Beckman LS 3801) for 10 min to quantify the 14C in
each fraction.
| |
RESULTS |
Reduction of TNT catalyzed by CODH.
Experiments were conducted
to determine whether purified CODH catalyzed the CO-dependent reduction
of TNT in the absence of electron transfer mediators such as methyl
viologen or ferredoxins. In these studies, catalytic amounts of CODH
were added to buffered solutions containing TNT in the presence of 1 atm of CO. Samples were taken periodically and analyzed by HPLC. The
resulting chromatograms and spectra are shown in Fig. 1. Prior to
adding CODH, the only species detected was TNT (peak A in the
t = 0 chromatogram). After 2.5 min, virtually all TNT
was consumed and two other species appeared (peaks B and C in the
t = 2.5 min chromatogram). The combined concentration
of these species increased at roughly the same rate as the TNT declined
(not shown). The UV-VIS spectra of B and C (Fig.
1, right panel) and their HPLC retention
times matched those of 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT) and 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT) respectively, and we
tentatively identify the products as those previously characterized. At
a longer reaction time (Fig. 1, t = 175 mm), these
monohydroxylamino derivatives were absent, and species D was present at
its highest concentration. The HPLC retention time and UV-VIS spectrum
of D were identical to 2,4-dihydroxylamino-6-nitrotoluene (24DHANT). At
even longer times (Fig. 1, t = 24 h), 24DHANT converted
to more polar species (labeled E). The retention time and UV-VIS spectrum of E matched the final product of TNT transformation by a C. acetobutylicum extract, which was reported
previously (7). This product, either
4-amino-6-hydroxylamino-3-methyl-2-nitrophenol or
6-amino-4-hydroxylamino-3-methyl-2-nitrophenol, results from a
catalyzed Bamberger rearrangement of 24DHANT (7).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
HPLC chromatogram (left panel) and UV-VIS spectra (right
panel) of CODH-dependent TNT degradation products. CODH (3.5 µg) was
injected into a vial containing 440 µM TNT and 1 atm of CO at room
temperature. Aliquots (100 µl) were analyzed by HPLC at
t = 0, 0.042, 2.9, and 24 h after injection and
then monitored at 230 nm. UV-VIS spectra were obtained for peak
fractions, designated A to E. Acid was not used to quench the
reaction.
|
|
Similar experiments were repeated in the absence of either CO or CODH.
TNT was not reduced by CO in the absence of CODH nor
by CODH in the
absence of CO. Moreover, the species B, C, D, and
E were stable for at
least 1 day under anaerobic conditions in
the absence of CO or CODH. We
conclude that CODH catalyzes the
CO-dependent reduction of TNT to
2HA46DNT and 4HA26DNT and catalyzes
the reduction of these compounds to
24DHANT.
Kinetics of the first step in the CODH-catalyzed TNT
transformation.
The CODH-catalyzed rate of TNT degradation to
2HA46DNT and 4HA26DNT at various concentrations of TNT is shown in Fig.
2 (left panel). Inverse rates in the
linear range (the first 60 s) were plotted as a function of
inverse substrate concentration in Fig. 2 (right panel). CODH-catalyzed
degradation of TNT exhibited saturation kinetics, indicating that TNT
bound to one of the two active sites of CODH. The apparent
Km for CODH for TNT was 165 ± 43 µM and the kcat was 400 ± 94 s1
under the conditions of the experiment.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2.
Kinetics of CODH-dependent TNT degradation. CODH (1.8 µg) was injected into a vial at room temperature containing 1 atm of
CO and 41 ( ), 62 ( ), 83 ( ), 109 ( ), and 210 ( ) µM TNT.
(Left panel) Plot of TNT concentration versus time after adding CODH.
(Right panel) Double-reciprocal plot using initial velocities (0 to
60 s) from the left-panel data. The line was curve fitted by
linear regression with 95% confidence. The standard deviation values
for the kinetic parameters were determined by using StatMost
Software.
|
|
Time course of TNT transformation and product formation.
To
monitor the pathway of CODH-catalyzed TNT transformation,
14C-labeled TNT was employed to determine the proportion of
each intermediate at various times during the reaction. Results from samples removed at various times and monitored by HPLC are plotted in
Fig. 3 as the percentage of the
14C (initially present as TNT) in various intermediates as
a function of time after adding CODH. The enzyme quickly converted TNT
to 4HA26DNT and 2HA46DNT, with about four times more 4HA26DNT produced than 2HA46DNT. These monohydroxylamino derivatives slowly
converted to 24DHANT, which eventually converted to the Bamberger
rearrangement product.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Temporal transformation of TNT by CODH. The initial
compound TNT () was converted to 4HADNT (), 2HADNT (),
24-DHANT (), and polar product resulting from Bamberger
rearrangement () during the reaction with CODH in the presence of
saturated CO. For reasons described in Materials and Methods, the
enzyme amount (1.8 µg) used for the fast early phase of TNT
transformation was 1/60th of that used for the latter, slower phase of
TNT transformation. The reaction time is normalized for the same amount
of enzyme (1.8 µg).
|
|
Inhibition of CODH-catalyzed transformation of TNT by cyanide.
Cyanide is a potent inhibitor of the CO oxidation activity of CODH.
Therefore, experiments were conducted to examine the impact of
CN on CODH-catalyzed transformation of TNT. As shown in
Fig. 4, enzyme treated with
CN was inactive toward TNT, while the control sample
rapidly transformed TNT. One peculiar (and poorly understood) property
of CN-inhibited enzyme is that it can be reactivated by
incubation in an atm of CO for ca. 1 h (1).
CN-inhibited enzyme was reactivated to some extent (13%)
toward TNT degradation after incubating 40 min under 1 atm of CO.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 4.
Analysis of inhibition of CODH-catalyzed TNT
degradation. The degradation of TNT with enzyme treated with CN ()
or left untreated () is shown. CODH (1.8 µg) was treated with 10 eq/  KCN for about 0.5 h and then assayed as described in the
text in 2 ml of assay buffer (lacking cyanide) with 220 µM TNT and 1 atm of CO. An equivalent sample not treated with KCN was also
assayed.
|
|
| |
DISCUSSION |
Our results show that in the absence of electron transfer
mediators, the purified CODH catalyzes the reductive degradation of
TNT. TNT is rapidly reduced to the initial intermediates
4-hydroxylamino-2,6-dinitrotoluene (~80%) and
2-hydroxylamino-4,6-dinitrotoluene (~20%). CODH then catalyzes
the reductive transformation of these monohydroxylamino derivatives, at
a slower rate than the initial reduction of TNT, to
2,4-dihydroxylamino-6-nitrotoluene. The final product of CODH transformation is a Bamberger rearrangement product. Moreover, CODH-catalyzed TNT transformation exhibited saturation kinetics, indicating that TNT binds and is reduced at one of the two active sites
on the enzyme. In a previous report, partially purified CODH reduced
2,4-diamino-6-nitrotoluene (DANT) to 2,4-diamino-6-hydroxylaminotoluene in the presence of methyl viologen and CO (10). The
catalytic rate of CODH-catalyzed DANT reduction reported in that study
was estimated to be about 8 s1. However, it was not
determined whether the reduction was catalyzed by CODH or if methyl
viologen was responsible for the reduction and CODH simply served to
reduce methyl viologen. Since the reduction of DANT also occurred in
the presence of dithionite-reduced methyl viologen (or benzyl viologen
or ferredoxin) and in the absence of CODH, the reduction was concluded
to be nonspecific and probably mediated by any redox enzyme capable of
reducing viologens or ferredoxins (10). In contrast, our
results indicate a catalytic rate of 400 s1 for TNT
transformation and suggest that TNT binds to an active site of CODH.
Furthermore, CODH was capable of reducing aryl nitro groups in the
absence of auxiliary electron carriers.
The fact that cyanide inhibits the reaction and that adding CO restores
activity indicates a CO activated site is required for TNT reduction
and suggests that TNT may bind to the active site for
CO-CO2 redox catalysis, namely, the C cluster. Given the
electronic similarities of CO2 and an aryl nitro group
(R-NO2) and the proposed mechanism for the two-electron
reduction of CO2 by CODH (2, 15), it is
reasonable to suggest a related mechanism for the four-electron
reduction of TNT. This mechanism would involve a nucleophilic attack
and two-electron reduction of R-NO2 by the fully reduced
(Cred2) state of the enzyme, followed by two protonations and loss of water. The resulting nitroso intermediate (RNO) would be
similarly attacked and reduced by another fully reduced enzyme, and two
more protonations would yield the product RNHOH. Further studies are
required to test the validity of this mechanism.
The reaction intermediates and products of TNT reported here appear to
be identical to those found in the transformation of TNT by C. acetobutylicum (7, 8), suggesting that this organism may contain CODH or an enzyme exhibiting a similar mechanism. A CODH
has been reported in C. pasteurianum (18), and
elements of CODH have been found in a BLAST search of the genome of
C. acetobutylicum ATCC 824. However, CODH may not be
responsible for TNT degradation in C. acetobutylicum since
transformation was stimulated by H2 (8). Further
comparison of TNT transformation in these two different clostridia
would give insights into the enzymatic mechanisms of TNT transformation
in anaerobic bacteria. Information gained from this study will help
identify optimal conditions and point out potential limitations or
opportunities of particular enzymatic systems for the degradation of explosives.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the DSWA project
01-97-1-0020 and by the Advanced Technology Program of Texas (FICE code
010366, project 020).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Cell Biology, MS-140, Rice University, 6100 Main St., Houston, TX 77005-1892. Phone: (713) 348-4920. Fax: (713) 348-5154. E-mail: gbennett{at}bioc.rice.edu.
| |
REFERENCES |
| 1.
|
Anderson, M. E., and P. A. Lindahl.
1994.
Organization of clusters and internal electron pathways in CO dehydrogenase from Clostridium thermoaceticum: relevance to the mechanism of catalysis and cyanide inhibition.
Biochemistry
33:8702-8711[CrossRef][Medline].
|
| 2.
|
Anderson, M. E., and P. A. Lindahl.
1996.
Spectroscopic states of the CO oxidation/CO2 reduction active site of carbon monoxide dehydrogenase and mechanistic implications.
Biochemistry
35:8371-8380[CrossRef][Medline].
|
| 3.
|
Bryant, C., and M. DeLuca.
1991.
Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae.
J. Biol. Chem.
266:4119-4125[Abstract/Free Full Text].
|
| 4.
|
DeRose, V. J.,
J. Telser,
M. E. Anderson,
P. A. Lindahl, and B. M. Hoffman.
1998.
A multinuclear ENDOR study of the C-cluster in CO dehydrogenase from Clostridium thermoaceticum: evidence for HxO and histidine coordination to the [Fe4S4] center.
J. Am. Chem. Soc.
120:8767-8776[CrossRef].
|
| 5.
|
Hu, X.,
N. J. Spangler,
M. E. Anderson,
J. Xia,
P. W. Ludden,
P. A. Lindahl, and E. Münck.
1996.
Nature of the C-cluster in Ni-containing carbon monoxide dehydrogenase.
J. Am. Chem. Soc.
118:830-845[CrossRef].
|
| 6.
|
Hughes, J.,
C. Y. Wang,
R. Bhadra,
A. Richardson,
G. Bennett, and F. Rudolph.
1998.
Reduction of 2,4,6-trinitrotoluene by Clostridium acetobutylicum through hydroxylamino-nitrotoluene intermediates.
Environ. Toxicol. Chem.
17:343-348[CrossRef].
|
| 7.
|
Hughes, J.,
C. Wang,
K. Yesland,
A. Richardson,
R. Bhadra,
G. Bennett, and F. Rudolph.
1998.
Bamberger rearrangement during TNT metabolism by Clostridium acetobutylicum.
Environ. Sci. Technol.
32:494-500[CrossRef].
|
| 8.
|
Khan, T. A.,
R. Bhadra, and J. Hughes.
1997.
Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum.
J. Ind. Microbiol. Biotechnol.
18:198-203[CrossRef].
|
| 9.
|
McCormick, N. G.,
F. E. Feeherry, and H. S. Levinson.
1976.
Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.
Appl. Environ. Microbiol.
31:949-958[Abstract/Free Full Text].
|
| 10.
|
Preuss, A.,
J. Fimpel, and G. Diekert.
1993.
Anaerobic transformation of 2,4,6-trinitrotoluene (TNT).
Arch. Microbiol.
159:345-353[CrossRef][Medline].
|
| 11.
|
Ragsdale, S. W.
1991.
Enzymology of the acetyl-CoA pathway of CO2 fixation.
CRC Crit. Rev. Biochem. Mol. Biol.
26:261-300.
|
| 12.
|
Ragsdale, S. W.,
J. R. Baur, and P. A. Lindahl.
1991.
The amino acid sequence and redox properties of the 8-iron ferredoxin II from Clostridium thermoaceticum.
FASEB J.
5:A470-A470.
|
| 13.
|
Ragsdale, S. W., and M. Kumar.
1996.
Nickel-containing carbon monoxide dehydrogenase/acetyl-CoA synthase.
Chem. Rev.
96:2515-2539[CrossRef][Medline].
|
| 14.
|
Russell, W. K.,
M. V. Stülhandske,
J. Xia,
R. A. Scott, and P. A. Lindahl.
1998.
Spectroscopic, redox, and structural characterization of the Ni-labile and nonlabile forms of the acetyl-CoA synthase active site of carbon monoxide dehydrogenase.
J. Am. Chem. Soc.
120:7502-7510[CrossRef].
|
| 15.
|
Seravalli, J.,
M. Kumar,
W.-P. Lu, and S. W. Ragsdale.
1997.
Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: kinetic characterization of the intermediates.
Biochemistry
36:11241-11251[CrossRef][Medline].
|
| 16.
|
Schackmann, A., and R. Müller.
1991.
Reduction of nitroaromatic compounds by different Pseudomonas species under aerobic conditions.
Appl. Microbiol. Biotechnol.
34:809-813.
|
| 17.
|
Shin, W.,
M. E. Anderson, and P. A. Lindahl.
1993.
Heterogenous nickel environments in carbon monoxide dehydrogenase from Clostridium thermoaceticum.
J. Am. Chem. Soc.
115:5522-5526[CrossRef].
|
| 18.
|
Thauer, R. K.,
G. Fuchs,
B. Käufer, and U. Schnitker.
1974.
Carbon monoxide oxidation in cell-free extracts of Clostridium pasteurianum.
Eur. J. Biochem.
45:343-349[Medline].
|
| 19.
|
Wang, C., and J. B. Hughes.
1998.
Derivatization and separation of 2,4,6-trinitrotoluene metabolic products.
Biotechnol. Tech.
12:839-842[CrossRef].
|
| 20.
|
Xia, J., and P. A. Lindahl.
1996.
Assembly of an exchange-coupled [Ni:Fe4S4] cluster in the subunit of carbon monoxide dehydrogenase from Clostridium thermoaceticum with spectroscopic properties and CO-binding ability mimicking those of the acetyl-CoA synthase active site.
J. Am. Chem. Soc.
118:483-483[CrossRef].
|
| 21.
|
Xia, J.,
Z. Hu,
C. V. Popescu,
P. A. Lindahl, and E. Münck.
1997.
Mössbauer and EPR study of the Ni-activated -subunit of carbon monoxide dehydrogenase from Clostridium thermoaceticum.
J. Am. Chem. Soc.
119:8301-8312[CrossRef].
|
| 22.
|
Zucker, M., and A. Nason.
1955.
Nitroaryl reductase from Neurospora crassa.
Methods Enzymol.
2:406-411[CrossRef].
|
Applied and Environmental Microbiology, April 2000, p. 1474-1478, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ziganshin, A. M., Gerlach, R., Borch, T., Naumov, A. V., Naumova, R. P.
(2007). Production of Eight Different Hydride Complexes and Nitrite Release from 2,4,6-Trinitrotoluene by Yarrowia lipolytica. Appl. Environ. Microbiol.
73: 7898-7905
[Abstract]
[Full Text]
-
Stenuit, B., Eyers, L., Rozenberg, R., Habib-Jiwan, J.-L., Agathos, S. N.
(2006). Aerobic Growth of Escherichia coli with 2,4,6-Trinitrotoluene (TNT) as the Sole Nitrogen Source and Evidence of TNT Denitration by Whole Cells and Cell-Free Extracts. Appl. Environ. Microbiol.
72: 7945-7948
[Abstract]
[Full Text]
-
Watrous, M. M., Clark, S., Kutty, R., Huang, S., Rudolph, F. B., Hughes, J. B., Bennett, G. N.
(2003). 2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum. Appl. Environ. Microbiol.
69: 1542-1547
[Abstract]
[Full Text]
-
Riefler, R. G., Smets, B. F.
(2002). NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction. Appl. Environ. Microbiol.
68: 1690-1696
[Abstract]
[Full Text]
-
Esteve-Nunez, A., Caballero, A., Ramos, J. L.
(2001). Biological Degradation of 2,4,6-Trinitrotoluene. Microbiol. Mol. Biol. Rev.
65: 335-352
[Abstract]
[Full Text]
-
Pak, J. W., Knoke, K. L., Noguera, D. R., Fox, B. G., Chambliss, G. H.
(2000). Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C. Appl. Environ. Microbiol.
66: 4742-4750
[Abstract]
[Full Text]
Top
Abstract
Introduction
