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J. Biol. Chem., Vol. 276, Issue 44, 40991-40997, November 2, 2001
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From the
Received for publication, July 3, 2001, and in revised form, August 23, 2001
The United Nations and the U.S. Environmental
Protection Agency have identified a variety of chlorinated aromatics
that constitute a significant health and environmental risk as
"priority organic pollutants," the so-called "dirty dozen."
Microbes have evolved the ability to utilize chlorinated aromatics as
terminal electron acceptors in an energy-generating process called
dehalorespiration. In this process, a reductive dehalogenase (CprA),
couples the oxidation of an electron donor to the reductive elimination
of chloride. We have characterized the B12 and
iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive
dehalogenase from Desulfitobacterium chlororespirans. By
defining the substrate and inhibitor specificity for the dehalogenase,
the enzyme was found to require an hydroxyl group
ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group
can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to
be eliminated. The purified D. chlororespirans enzyme could
dechlorinate an hydroxylated PCB
(3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that
with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect
studies indicate that transfer of a single proton is partially
rate-limiting in the dehalogenation reaction.
Associated with the United Nations Environmental Program, in May
2001, a global treaty has been adopted that aims to eliminate 12 persistent organic pollutants, the so-called "dirty dozen," which
are chemicals that pose a significant risk to health and the
environment. Eleven of the twelve targeted compounds are chlorinated organic compounds. A U.S. Environmental Protection Agency list of its
12 priority Persistent, Bioaccumulative, and Toxic pollutants includes
nine chlorinated organics, five of which are chlorinated aromatics.
Bioremediation represents one possible strategy for removing these
environmental toxins. A variety of microbes can catalyze the removal of
the halogen substituent (1, 2). These organisms and the dehalogenation
reactions they catalyze are interesting from the standpoint of
biochemistry, genetics, and biotechnological applications.
Dehalogenation reactions are also intriguing from an evolutionary
perspective since chlorinated xenobiotics were introduced within
the last century.
There are at least three distinct enzyme classes that can remove the
halogen substituent from organic compounds: the hydrolases, the
methyltransferases, and the reductive dehalogenases. In this report we
focus on an enzyme in the reductive dehalogenase class. The hydrolytic
dehalogenases, which replace the chloro group with an hydroxyl
functionality, are by far the best studied. Hallmarks of this enzyme
class are the use of an aspartate residue as a nucleophile to form a
covalent enzyme-substrate adduct and a general base residue to
facilitate the hydrolysis step. High resolution structures are
available for the 4-chlorobenzoyl-CoA (3), haloacid (4), and
haloalkane (5) dehalogenases. The methyltransferase class uses
vitamin B12 as a cofactor in a group transfer reaction (6).
The reductive dehalogenases replace the chloro functionality with a
hydride equivalent. Two types of reductive dehalogenases include
dehalorespiration-linked metalloenzymes that couple the reduction of
the chlorinated hydrocarbons to ATP synthesis via a chemiosmotic
mechanism (2, 7) and members of the glutathione S-transferase class, which feature a
glutathione-dependent substitution reaction in their
mechanism (8).
This report focuses on a dehalorespiration-linked reductive
dehalogenase. Members of the dehalorespiratory class of reductive dehalogenases have several interesting features. Enzymes have been
described that can dehalogenate a discrete set of aliphatic and
aromatic compounds. Perhaps the most interesting and potentially important reactions are the microbial reductive dechlorination of
polychlorinated biphenyls
(PCBs)1 and their oxygenated
metabolites, the hydroxylated PCBs (OH-PCBs), by anaerobes (9, 10) like
Desulfitobacterium dehalogenans (11). Microbes have been
isolated from contaminated soils and sediments that can catalyze
removal of meta, para, and ortho
chlorines of PCB mixtures by reductive dehalogenation (12).
Furthermore, some microbes have evolved the capacity to gain energy
from the reductive dehalogenation reaction (13, 14). For example,
Dehalospirillum multivorans generates a membrane potential
and/or a pH gradient associated with the transfer of electrons from the
electron donor (H2 or pyruvate) to the chlorinated organic
(15). In all but one case, the enzymes, encoded by the cprA
gene, that catalyze the reductive dehalogenation are vitamin
B12- (or cobamide-) and iron-sulfur-containing enzymes. In
at least some desulfitobacteria, the cprA gene is found
within a cluster of genes that includes a membrane anchor (CprB), a
membrane-associated regulatory protein (CprC), a DNA binding protein
(CprK), and several chaperones (CprT, CprD, and CprB) (16). In this
report we will typically refer to the dehalogenase as CprA.
Two general mechanisms have been proposed for the dehalorespiring
reductive dehalogenases (modified from Ref. 2) (see Fig. 1). In one
(shown as path A), an organocobalt adduct is formed as in the
methyltransferases; whereas, in the other (path B), the corrinoid
serves as an electron donor. In path A, the halide is eliminated as the
aryl-Co(III) intermediate is formed. Path B resembles the Birch
reduction of hydroxylated aromatics in which a radical anion is formed.
Because the chlorine is the most electron-withdrawing substituent on
the ring, the carbon to which it is bonded would bear the highest
charge density in the radical anion. This position, therefore, would be
most activated toward protonation, assuming that the proton transfer
occurs before or concerted with chloride elimination, as shown in the
upper transition state diagram in Fig.
1. Yet one could imagine proton addition
from solvent occurring after chloride departure, as in the lower
transition state structure.
Because dehalorespiring bacteria were discovered only recently, many
questions about the dehalogenation mechanism remain unanswered. Are
organocobalt adducts analogous to those in
B12-dependent methyltransferases formed during
the reaction mechanism? How is the chlorine replaced by a hydride
equivalent? Does the reaction involve radical intermediates? What are
the structural requirements for the substrate of the dehalogenase
reaction? Here we describe studies focused mainly on characterizing the
substrate specificity and mechanism of the aromatic dehalogenase from
Desulfitobacterium chlororespirans, an organism that was
enriched and isolated based on its ability to grow on
3-chloro-4-hydroxybenzoate (17). These studies are important in
understanding the dehalogenation mechanism and in developing strategies
for bioremediation of sites contaminated with chlorinated aromatics,
like PCBs.
Organism and Growth Conditions--
D.
chlororespirans (ATCC 700175) was grown with agitation on reduced
anaerobic medium (ATCC 2035), containing 20 mM pyruvate, 2 mM 3-chloro-4-hydroxybenzoate, and 1 g of yeast
extract per liter (17). The 13-liter cultures were incubated in a
14-liter fermentor at 37 °C for 4 days using an 80%
N2/20% CO2 headspace gas. Addition of 20 mM pyruvate and 2 mM 3-chloro-4-hydroxybenzoate on the second day of incubation increased the optical density at 600 nm
(A600) to 0.7-0.8. D. chlororespirans cells were harvested with a continuous flow
CEPA centrifuge at 16,000 × g, yielding 1.4 g/liter wet weight of cells, which were stored at Purification of CprA--
14.6 g of cells was suspended in 35 ml
of buffer A (100 mM potassium Pi, pH 7.2, 2 mM dithiothreitol) plus 40 units/ml DNase I and sonicated
(30 min, 30 s pulse on, 30 s pulse off) under anaerobic
conditions (95% N2/5% H2). The cell-free
extract was separated into a membrane fraction and a soluble fraction
by centrifugation for 4 h at 105,000 × g and
4 °C. The membrane fraction was resuspended in 20 ml of buffer A
supplemented with 3% (w/v) Triton X-100 and 20% glycerol and
incubated at 4 °C for 2 h under anaerobic conditions. The
insoluble fraction was removed from this preparation by centrifugation for 2 h at 105,000 × g and 4 °C. The
supernatant contained CprA, which was further processed.
All chromatographic steps were performed in an anaerobic chamber with a
N2/H2 (95%/5%) gas phase. The membrane
fraction was diluted with an equal volume of buffer B (50 mM potassium Pi, 0.1% Triton X-100, 20%
glycerol, and 2 mM dithiothreitol) at pH 6.2 and loaded on
a 5 × 7 cm Q-Sepharose column (Bio-Rad) equilibrated with the
same buffer. The column was eluted with a 600-ml linear gradient from 0 to 500 mM NaCl in buffer B at a flow of 3 ml/min.
Fractions containing the highest dechlorination activity were pooled
and diluted with an equal volume of buffer B (same as buffer A, but at
pH 8.0) and applied to a High Q-Sepharose column (2.5 × 7 cm)
(Bio-Rad) equilibrated with buffer B. The enzyme was eluted with a
120-ml linear gradient from 0 to 500 mM NaCl in buffer B at
a flow rate of 1.3 ml/min.
Fractions containing dehalogenase activity were pooled and mixed with
an equal volume of buffer B, pH 8.0, and applied to a High Q-Sepharose
column (Econo-Pac cartridges, 5 ml) (Bio-Rad) equilibrated with the
same buffer. The enzyme was eluted with a 60-ml linear gradient from 0 to 600 mM NaCl in buffer B at a flow rate of 1 ml/min.
The protein concentration was determined according to Bradford (18)
with bovine serum albumin as a standard. The pH and temperature optima
were determined in buffer B at pH values ranging from 5.1 to 8.7 and
temperatures from 25 to 70 °C.
Dehalogenase activity in every fraction was assayed by TLC, as
described below. Dehalogenase activity in the combined fractions after
each step of purification was estimated spectrophotometrically, as
described below.
TLC--
The assay mixture, which contained 0.3 ml of buffer B
(pH 7.6), 40 µl of 15 mM methyl viologen, 50 µl of 0.2 M titanium citrate, 10 µl of 50 mM
3-chloro-4-hydroxybenzoate, and 10-100 µl of sample, was
anaerobically incubated for 30 min at 57 °C and then quenched by
adding 50 µl of 0.2 M perchloric acid and centrifuged at
10,000 × g for 1 min. The clear supernatant was
extracted with 0.5 ml of diethyl ether, and the organic phase was
spotted on Silica Gel AL SIL G/UV plates (Whatman Ltd.). The mobile
phase consisted of toluene:acetic acid:water (6:7:3, v/v). The
separated products were visualized by use of a UV lamp and identified
by their relative mobility (Rf) values, which
are 0.46 and 0.32 for 3-chloro-4-hydroxybenzoate and 4-hydroxybenzoate, respectively.
Spectrophotometric Dehalogenase Assay and Determination of
Kinetic Parameters--
Dehalogenation of 3-chloro-4-hydroxybenzoate
and other chlorinated substrates was measured spectrophotometrically at
578 nm and 37 °C by following the oxidation of reduced methyl
viologen ( Measuring Dehalogenation of a Hydroxy-PCB--
In studies of the
dehalogenation of hydroxy-PCBs, the 0.5-ml assay mixture contained 20 mM titanium(III) citrate, 0.6 mM
3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 22-28 µg/ml
dehalogenase in 50 mM Tris-HCl buffer, pH 7.6. After
incubating at 57 °C for various times, the reaction was quenched by
exposing to air and extracting with dichloromethane. The organic phase
was analyzed for the parent compound
(3,3',5,5'-tetrachloro-4,4'-biphenyldiol) and dechlorination products
by gas chromatography-mass spectrometry (GC-MS). Injections (1 µl)
were made in the splitless mode on a Varian 3400CX GC coupled to a
Saturn 3 ion trap mass spectrometer using an 8200 Autosampler and
helium as the carrier gas. A fused silica GC column (30 ml × 0.25 mm i.d.) coated with 0.25-µm DB-XLB (Agilent Technologies/J&W
Scientific Division, Folsom, CA) was used to separate compounds of
interest. The GC was programmed as follows: 70 °C (2-min hold); ramp
to 280 °C at 6 °C/min (3-min hold); total run time of 40 min. The
injector was initially held at 70 °C and then ramped ballistically
to 280 °C at 200 °C/min. The MS transfer line and manifold
temperature were held isothermal at 280 °C and 240 °C,
respectively. The ion trap was operated in the electron impact full
scan mode (m/z 50-350) at 1.0 cycles/s and was
turned on 8 min after sample injection. Repeated injections of a 100 µg/ml solution of the parent 3,3',5,5'-tetrachloro-4,4'-biphenyldiol (m/z 223, 322, 324) were made to determine the
electron impact mass spectrum and to assess the response and the change
in GC-MS sensitivity, if any, through the analytical run. No
variability in relative response and/or mass spectra of parent/daughter
compounds was observed. Two additional GC peaks that appeared to be
related to the parent compound were detectable in all extracts,
including a trichloro-biphenyldiol (m/z 189, 288, 290) and a dichloro-biphenylol (m/z 139, 238, 240). The relative abundances and molar concentrations of these
dichloro- to tetrachloro- compounds were estimated assuming a uniform
MS response equal to that determined for the parent compound and the
appropriate molecular masses. The purity of the parent
3,3',5,5'-tetrachloro-4,4'-biphenyldiol was reported by the supplier to
be 95%. We estimated the purity at 92%, with as much as 7%
trichloro-biphenyldiol as impurity. Less than 1% of the
dichloro-biphenyl-ol and no dichloro-biphenyldiol was found in the
original solution. The mean relative amounts of the
parent/dechlorination products were 50%/50% for incubations
containing the dehalogenase compared with 77%/23% for duplicate
control incubations lacking the enzyme.
Analyses of the Chlorophenol Dehalogenase--
The apparent
molecular mass of the purified enzyme was determined by 12%
SDS-polyacrylamide gel electrophoresis according to Laemmli (20). A low
molecular weight marker kit (Bio-Rad) was used as a reference. The gels
were stained with Coomassie Brilliant Blue G-250. The corrinoid content
of CprA was estimated from the absorption spectrum of the dicyano
derivative using an extinction coefficient,
To determine the N-terminal amino acid sequence, purified enzyme was
transferred from a 10% SDS-polyacrylamide gel onto a polyvinylidene
difluoride membrane (Immobilon polyvinylidene difluoride, Millipore
Corp.) by blotting with a Trans-Blot SD semidry transferring cell
(Bio-Rad). Blotting was carried out at 36 V for 1.5 h using a
transfer buffer containing 20 mM Tris, 150 mM
glycine, and 20% methanol, pH 9.1. The transferred protein band was
stained with 0.1% Amido black, excised, and sequenced by automated
N-terminal Edman degradation using an ABI-494 Procise sequencer.
Purification of CprA--
The 3-chloro-4-hydroxybenzoate reductive
dehalogenase (CprA) was purified from D. chlororespirans
cells that were grown on pyruvate as a carbon and electron
source and 3-chloro-4-hydroxybenzoate as an electron acceptor. More
than 90% of the enzyme activity was found in the membrane fraction and
was extracted with the nonionic detergent Triton X-100 (3% v/v) (Table
I). The tetrachloroethylene dehalogenase
from Desulfitobacterium strain PCE-S (7) and the 3-chloro-4-hydroxyphenylacetate reductive dehalogenase from
Desulfitobacterium hafniense (23) are also
membrane-associated and must be extracted and purified in the presence
of detergent.
The purification scheme for 3-chloro-4-hydroxybenzoate dehalogenase is
summarized in Table I. During purification, the specific activity
increased 181-fold to a final value of 15.4 units/mg of protein.
SDS-gel electrophoresis analysis of purified dehalogenase revealed a
single band of ~50-kDa molecular mass (Fig.
2). The requirement for detergent to
stabilize the protein prevents an accurate determination of the native
molecular mass. For example, the size of the monomeric PCE reductive
dehalogenase from D. strain PCE-S was determined by
gel filtration to be 198 kDa, which is more than three times higher
than the apparent mass estimated by SDS-page electrophoresis (65 kDa)
(24). Similar results were obtained for the PCE reductive dehalogenase
from Dehalobacter restrictus (25). As shown by gel
filtration and ultracentrifugation studies of heterodisulfide
reductase, which is also isolated in the presence of Triton, much of
the molecular mass is contributed by tightly associated detergent
(26).
Properties of CprA--
The N-terminal amino acid sequence of CprA
is AATDTLNYVPGRKKQNSKLL. This sequence is similar to those of
dehalogenases from other Desulfitobacteria, e.g.
D. hafniense (55%), D. dehalogenans (45%),
D. viet-1 (45%), and D. PCE-1 (45%) (Fig.
3). Because the first amino acid is Ala,
the protein probably has been post-translationally processed as have
the other dehalogenases, which contain a 40-amino acid signal sequence
that is removed as the protein matures and binds to the bacterial
membrane (27). All the published sequences of the reductive
dehalogenases that correspond to an active protein from
Desulfitobacterium strains contain a conserved NYVPG
sequence (Fig. 3).
After treating the 3-chloro-4-hydroxybenzoate reductive dehalogenase
with potassium cyanide at 90 °C and removing the protein precipitate, the typical visible light absorption spectrum of dicyano-cob(III)alamin (21) is observed (inset in Fig.
4). Using an extinction coefficient at
580 nm of 10.13 mM
The D. chlororespirans enzyme is completely inhibited when
the enzyme is incubated with 450 µM propyl iodide.
Approximately 50% of its initial activity is regained when the enzyme
is exposed to light. This characteristic of many cobamide-containing
proteins provides evidence for an important role of the corrinoid in
the dehalogenation reaction. The dehalogenase activity is 90%
inhibited by incubation with 45 mM KCN and completely
inhibited by 4 mM sodium sulfite or 7 mM EDTA.
Sodium citrate had no influence on the dehalogenation activity at
concentrations up to 170 mM.
The UV-visible spectrum of the native enzyme has a broad absorption
maximum between 410 and 450 nm and a shoulder at 310 nm (Fig. 4). This
spectrum is reminiscent of the corrinoid iron-sulfur protein from
acetogens (29) and methanogens (30). The absorption maximum at 410 nm,
which is assigned to the iron-sulfur clusters, is superimposed on the
absorbance in the 450- to 470-nm region, which is characteristic of
cobalamin in the 2+ oxidation state. The lack of absorption peaks at
385-390 nm or at 360 nm indicates that there is little cob(I)alamin or
cob(III)alamin, respectively, in the as-isolated dehalogenase, which is
a characteristic of many corrinoid proteins (31). Because the
Co(II)/(I) couple is generally between Kinetic Properties of the D. chlororespirans Dehalogenase--
In
contrast to the PCE reductive dehalogenase from D. multivorans, which can be effectively reduced with titanium(III)
citrate (33), a redox mediator is required to observe catalysis by the D. chlororespirans enzyme. No dehalogenation was observed
with dithionite (up to 110 mM) or titanium(III) citrate (up
to 13 mM) alone as electron donors. Methyl viologen serves
as an effective redox dye. Dehalogenase activity is observed in crude
extracts of D. chlororespirans with or even without
titanium(III) citrate presumably due to the physiological electron
donor that is present in the extract (34).
The pH optimum of the dehalogenase is ~6.8 (not shown, Supplemental
Fig. 1S). The highest enzymatic activity was obtained at 59 °C (not
shown, Supplemental Fig. 2S), which is similar to the property of a
partially purified dehalogenase from D. chlororespirans (34). The oxygen sensitivity of the CprA (t1/2 = 77 min) is similar to those of the enzymes from D. dehalogenans (t1/2 = 90 min (35)), D. strain PCE-S (t1/2 = 50 min (24)) and the
membrane fraction of D. hafniense (t1/2 = 100 (23)). The only known oxygen-stable reductive dehalogenase is the
heme-containing enzyme from D. tiedjei (28).
Given the potential use of reductive dehalogenases in environmental
bioremediation, it is important to establish their substrate specificity. CprA exhibits Michaelis-Menten kinetics with a variety of
chlorinated phenols (Fig. 5 and Table
II). The substrate range of the
purified dehalogenase (Table II) agrees well with that reported for the
D. chlororespirans cells (17) and its membrane fraction (34), indicating that a single enzyme is involved in dehalogenation of chlorinated phenols by this bacterium. We
compared the specificity parameters
(kcat/Km) for different
chlorinated aromatics that can undergo dehalogenation, including
3-chloro-4-hydroxybenzoate, 3,5-chloro-4-hydroxybenzoate,
3-chloro-4-hydroxyphenylacetate, 2,3-dichlorophenol,
2.6-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol (Table
II). Lack of an hydroxyl group in the position ortho to the
chlorine substituent prevents dehalogenation. For example, 3- or
4-chlorobenzoate and 2,6-, 2,4-, or 3,5-dichlorobenzoate are not
metabolized. Chlorine does not replace the requirement for the
hydroxyl group, because 3,4-dichlorobenzoate is not dehalogenated.
Barring one exception, the enzyme only metabolizes aromatic compounds;
tetrachloroethene, trichloroethane, and 2,4-dichloropropene are not
dechlorinated. Trichloroacetate undergoes dehalogenation with a
specific activity of 18.8 µmol/mg/min, which is similar to that
observed with 3-chloro-4-hydroxybenzoate. Yet, unlike any of the
aromatic substrates, ~10% of the methyl viologen is consumed during
the reaction with trichloroacetate. One possible explanation for this
finding is that the viologen radical may react with and possibly form
an adduct with a radical intermediate either on the enzyme, as in
cobalamin-dependent methionine synthase (37), or the
substrate. Because this behavior is restricted to trichloroacetic acid,
it suggests that a substrate rather than an enzyme-based intermediate
reacts with the blue viologen cation radical. Further studies are
required to determine the reason for the consistent loss of viologen
when the enzyme utilizes trichloroacetate. Alternatively,
CprA-catalyzed dehalogenation of trichloroacetate follows a different
mechanism than that used for reduction of chlorinated phenols.
Why is an ortho hydroxyl group required for all the
chlorinated aromatic substrates? It could be required for binding or
could participate in the chemistry of the dehalogenation reaction. An hydroxyl group ortho to the halogen atom is required for the
tetrachlorohydroquinone reductive dehalogenase from Sphingomonas
chlorophenolica, which is a member of the glutathione
S-transferase superfamily (38, 39). In this case, the
ortho-hydroxyl group participates in elimination of an
enzyme-glutathione adduct that forms subsequent to the dechlorination
step.2
The requirement for an hydroxyl group ortho to the chlorine
that undergoes elimination represents a limitation for the
bioremediation of PCBs and related xenobiotics lacking an
ortho hydroxyl group. If the ortho-hydroxyl group
is involved in binding, such an hydroxyl group will also be required
for inhibitors of CprA activity. Consistent with the requirement for
binding, 4-hydroxybenzoate, the product of the
3-chloro-4-hydroxybenzoate dehalogenation reaction, and 3-hydroxybenzoate are competitive inhibitors (Ki = 1.7 and 12.8 mM, respectively)), whereas 3-chlorobenzoate
is not an inhibitor (Table III).
4-Hydroxybenzoate also inhibits the dehalogenation of
3-chloro-4-hydroxyphenylacetate and 2,4,6-trichlorophenol, with
similar Ki values (1.6 mM) as that of
3-chloro-4-hydroxybenzoate, which is consistent with competitive
inhibition. Similarly, 3,6-dichlorosalicylate (3,6-dichloro-2-hydroxybenzoate) inhibits the enzyme but
3,6-dichlorobenzoate and 3,6-dichloro-2-methoxybenzoate (dicamba) do
not.
Is the need for an hydroxyl group in the ortho position to
the chlorine a general attribute of corrinoid iron-sulfur-containing aromatic dehalogenases? This requirement has also been reported for the
enzymes that have been purified from D. dehalogenans (35) and D. hafniense (23). Yet, the heme-containing dehalogenase from D. tiedjei catalyzes meta dechlorination
(28). Because a major role of the hydroxyl group appears to be in
substrate binding, it is likely that corrinoid-dependent
enzymes containing modified substrate binding pockets will be found
that can catalyze dehalogenation with the hydroxyl group at other
positions of the ring and even lacking the hydroxyl group. D. hafniense (23, 40) and Desulfitobacterium
frappieri (41) cells can catalyze ortho,
meta, and para dehalogenation; however, the
corrinoid-containing dehalogenase from D. hafniense that
that has been purified apparently requires an ortho-hydroxyl
group (23) and none of the dehalogenases from D. frappieri
have been purified. Purification and characterization of dehalogenases
with different substrate specificities will help in understanding the
roles of corrinoid and of the hydroxyl group in the dehalogenation of
chlorinated phenols. Furthermore, because the major role for the
hydroxyl group is in binding, we surmise that it may be possible to
engineer a dehalogenase like the D. chlororespirans enzyme
that lacks this requirement.
With the D. chlororespirans dehalogenase, an hydroxyl group
ortho to the chlorine is necessary, but not sufficient for
binding to the enzyme, because 2-chlorophenol is not a substrate or
inhibitor (Km, Ki > 1 M). When there is a carboxyl or acetyl group
meta to the chlorine (3-chloro-4-hydroxybenzoate or
3-chloro-4-hydroxyphenylacetate), the compound is a substrate. Interestingly, placing another chlorine in the ortho or
meta position relative to the chlorine, is sufficient for
reactivity, albeit only 6.5 or 0.3% relative to the
para-carboxyl. The ortho-chloro derivative
(2,3-dichlorophenol) is approximately as reactive as the
meta-acetyl (3-chloro-4-hydroxyphenylacetate). However,
2,4-dichlorophenol is neither a substrate nor an inhibitor. Adding
additional halides to the ring only marginally enhances reactivity,
because the specificity factors for 2,4,6-trichlorophenol and
pentachlorophenol are only 20- and 1.3-fold higher, respectively, than
2,6-dichlorophenol. Even though 3,6-dichlorosalicylate
(3,6-dichloro-2-hydroxybenzoate) has an ortho hydroxyl and a
carboxyl group, it is not a substrate, but an inhibitor with a
Ki value (140 µM) 10-fold greater than
the Km for 3,5-dichloro-4-hydroxybenzoate (12 µM). The chloro group is less important for binding than
is the para-carboxyl or the ortho-hydroxyl,
because para- and meta-hydroxybenzoate are inhibitors.
Dehalogenation of Hydroxy-PCB--
The conditions for
dehalogenation of OH-PCBs and PCBs by anaerobic bacteria have been
reviewed previously (12). The ability of the D. chlororespirans dehalogenase to remove chlorine from aromatic
rings containing an ortho-hydroxyl group and another substituent in the position meta to the halide led us to
test whether hydroxylated polychlorinated biphenyls (OH-PCBs), which contain a meta-phenyl ring, can serve as substrates. The
OH-PCBs, formed by mono- and dioxygenases, are the main metabolites of PCBs. When CprA was incubated with
3,3',5,5'-tetrachloro-4,4'-biphenyldiol (tetrachlorobiphenyldiol) under
standard assay conditions, trichloro- and dichlorobiphenyldiol were
detected as the products (m/z 189, 288, 290 and
m/z 139, 238, 240, respectively) (Fig.
6a). There is increased
product formation with higher amounts of enzyme, demonstrating
enzyme-dependent degradation of the hydroxy-PCB. The rate
of this reaction in the presence of 0.6 mM substrate is
0.12 µmol/mg/min at 57 °C (Fig. 6b), which is ~0.3%
of the maximal rate with saturating levels of
3-chloro-4-hydroxybenzoate at the same temperature. This represents the
first example of OH-PCB dehalogenation by a pure enzyme and suggests
the possibility of using purified dehalogenases to aid in PCB
biodegradation. The requirement for the hydroxyl group for binding to
the enzyme assures that PCBs themselves will not serve as inhibitors.
However, OH-PCBs in most contaminated sites are a diverse mixture of
many congeners, some of which might inhibit and others which might react with the enzyme. No dehalogenation was observed with
2',3',4',5'-tetrachloro-3-biphenylol, 2',4',6'-trichloro-4-biphylol,
and 5-chloro-2-biphenylol, which is consistent with the requirement for
the chloro group to be ortho to the hydroxyl group.
2H2O Solvent Kinetic Isotope
Effects--
The kcat value for dehalogenation
of 3-chloro-4-hydroxybenzoate by the D. chlororespirans CprA
is 2.3-fold higher in 100% H2O than in 100%
2H2O (Fig. 7). A
fully rate-liming proton transfer would be expected to give a
2H2O solvent kinetic isotope effect (SKIE) of
~4; therefore, an intramolecular proton transfer reaction is
partially rate-limiting in the dehalogenase reaction. The linearity of
the proton inventory plot indicates that a single proton is in flight
in the transition state. These SKIE studies indicate that cleavage of
an H-O bond occurs in the rate-determining step of the dehalogenase
reaction. The haloalkane dehalogenase from Xanthobacter
autotrophicus GJ10 exhibits a 2H2O SKIE of
~1.9 on kcat; however, this enzyme catalyzes
replacement of the halide with hydroxide instead of "hydride." The
SKIE for haloalkane dehalogenase appears to originate from a slow
conformational change (42) in the enzyme associated with solvation of
the halide ion product as it leaves the active site (43). We suggest
that the SKIE for the reductive dehalogenase from D. chlororespirans might originate from the rate-limiting
deprotonation of a water molecule, because deuterium from solvent is
incorporated specifically into the carbon of 2,5-dichlorobenzoate that
undergoes dehalogenation by cell extracts of D. tiedjei
(44). A similar situation occurs in carbonic anhydrase, where
2H2O SKIEs between 2 and 3.8 are observed (36,
45-48). The SKIE studies seem most consistent with path B of Fig. 1,
in which the partially rate-limiting proton transfer step occurs after
an electron is transferred to the aromatic ring and before or concerted
with the departure of the chloride ion. It is not expected that
protonation of the neutral radical following homolysis would be
a rate-limiting step, as in path A. The SKIE could also derive from
solvation of the halide ion to facilitate elimination from the active
site, as has been proposed for the haloalkane dehalogenase (43).
Conclusion--
We have characterized the corrinoid- and
iron-sulfur-dependent reductive dehalogenase (CprA) from
D. chlororespirans. Steady-state kinetic studies of
substrate and inhibitor specificities indicate that the enzyme only
utilizes substrates with an hydroxyl group ortho to the
halide plus another functional group meta to the chlorine (carboxyl > acetyl > chloro). Because these
requirements appear to be mainly important for substrate binding, we
anticipate that enzymes can be designed or isolated that have the
ability to dehalogenate environmentally significant chloroaromatics.
The studies represent the first description of a purified dehalogenase that can catalyze the dechlorination of an hydroxylated PCB
(3,3',5,5'-tetrachloro-4,4'-biphenyldiol), albeit 100-fold slower than
the most reactive substrate tested so far. Solvent deuterium isotope
effect studies indicate that a single proton is in flight during a step
in the dehalogenation reaction that is partially rate-determining.
We thank Juergen Wiegel for help in early
stages of this project. We also thank Gabriele Diekert and Anke
Neumann for sharing the observation that reaction of PCE
dehalogenase with trichloroacetate consumes methyl viologen.
*
This work was supported by National Science Foundation Grant
MCB9974836 (to S. W. R).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE
68588-0664. Tel.: 402-472-2943; Fax: 402-472-7842; E-mail:
sragsdale1@unl.edu.
Published, JBC Papers in Press, August 31, 2001, DOI 10.1074/jbc.M106217200
2
S. D. Copley, personal communication.
The abbreviations used are:
PCB, polychlorinated biphenyl;
OH-PCB, hydroxylated PCB;
GC-MS, gas chromatography-mass spectrometry;
PCE, tetrachloroethene;
SKIE, solvent kinetic isotope effect.
Characterization of the B12- and
Iron-Sulfur-containing Reductive Dehalogenase from
Desulfitobacterium chlororespirans*,
,¶
Department of Biochemistry, Beadle Center,
University of Nebraska, Lincoln, Nebraska 68588-0664 and
§ Skidaway Institute of Oceanography, Savannah, Georgia
31411
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Mechanistic models for the corrinoid
iron-sulfur reductive dehalogenase. This was modified from a
previous study (2). The two pathways, A (dashed
arrows) and B (solid arrows), differ in the
roles of cobalamin. In A, an organocobalt adduct is formed;
in B, Co(I) donates an electron to the aromatic ring.
Possible transition states for a proton transfer reaction are shown in
the box at the upper right. Although the
mechanism and the transition state diagram assume a radical
intermediate, there is sparse evidence for it and there are other
viable possibilities. R, designates an additional functional
group on the ring.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C.
578 = 9.7 mM
1
cm1) (19). The 0.5-ml assay mixture contained 0.5 mM chlorinated compound and 0.3 mM methyl
viologen in buffer B at pH 7.6. The methyl viologen was reduced by
adding titanium(III) citrate until the absorbance at 578 nm reached
2.6. The linearity of the absorbance value to 2.6 OD units at 578 nm
was confirmed. The reaction was initiated by adding 5-50 µl of
dehalogenase (the final enzyme concentration was 20-150
nM). One unit of activity is defined as the amount of
enzyme that catalyzes the reduction of 1 µmol of chlorinated
substrate, which equals the oxidation of 2 µmol of reduced methyl
viologen, per minute. Steady-state kinetic parameters were determined
from plots of the initial rates of dehalogenation versus
substrate concentration (5 µM to 10 mM),
which were fit to the Michaelis-Menten equation using SigmaPlot. The
inhibition constant (Ki) was determined by fitting
experimental data to the following equation: v = Vmax*S/(S + Km*(1 + i/Ki)), where
Vmax and Km correspond to the parameters for 3-chloro-4-hydroxybenzoate dechlorination without inhibitor, and i stands for inhibitor concentration.
580, of
10.13 mM1 cm1 (21). A
48-element metal analysis was performed using the Jarrel-Ash 965 Atomcomp Plasma Emission Spectrograph at the Chemical Analysis Laboratory, University of Georgia (Athens, GA). Acid-labile sulfide was
determined according to a previous study (22).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Purification of 3-chloro-4-hydroxybenzoate reductive dehalogenase from
D. chlororespirans
View larger version (31K):
[in a new window]
Fig. 2.
12% SDS-polyacrylamide gel electrophoresis
with the purified 3-chloro-4-hydroxybenzoate reductive dehalogenase
from D. chlororespirans (7 µg,
lane 1). Lane 2 shows molecular size
markers. The gel was stained with Coomassie Brilliant Blue R-250.
View larger version (16K):
[in a new window]
Fig. 3.
Alignment of N-terminal amino sequences of
dehalogenases from D. chlororespirans
(1) and D. hafniense
(2) (23) and D. dehalogenans
(3) (35).
1 cm1 (21),
the corrinoid content is 0.84 ± 0.05 mol corrinoid/mol of
subunit. Besides cobalamin, a monomeric unit of the enzyme contains 7.6 g-atm iron and 1.0 g-atm Co, based on metal analysis, and 8.1 mol of
acid-labile sulfide. These results sugest that the dehalogenase from
D. chlororespirans contains one corrinoid and two
iron-sulfur clusters per monomeric unit. Except for the enzyme from
Desulfomonile tiedjei, which is apparently a heme enzyme
(28), the reductive dehalogenases so far purified from dehalorespiring
organisms contain cobamide and iron-sulfur clusters.
View larger version (15K):
[in a new window]
Fig. 4.
UV-visible absorption spectra of the purified
dehalogenase (0.15 mg/ml). Inset, dicyano-cob(III)alamin
absorption spectra after treating the 3 µM enzyme with 75 µM KCN at 90 °C.
500 and 600 mV and the
Co(III)/(II) couple is approximately +200 mV (32), most corrinoid
proteins isolated in the presence of mild reductants, like
dithiothreitol, are found in the Co(II) state.
View larger version (12K):
[in a new window]
Fig. 5.
Reductive dehalogenase activity as a function
of ( 
) 3-chloro-4-hydroxybenzoate and (
)
3-chloro-4-hydroxyphenylacetate concentrations. Enzyme activity
was monitored spectrophotometrically as described under "Experimental
Procedures." The data were fit to the Michaelis-Menten equation.
Kinetic parameters (Vmax, Km)
are listed in Table II.
Substrate specificity of reductive dehalogenase from D. chlororespirans
Inhibition of dehalogenase activity by chlorinated hydrocarbons
View larger version (11K):
[in a new window]
Fig. 6.
A, dependence of the trichlorodiol
formation rate on dehalogenase concentration. The concentration of
tetrachlorodiol was 0.6 mM, and the incubation time was 10 min. B, time dependence of trichlorodiol formation. The
concentrations of dehalogenase and tetrachlorodiol were 66 µg/ml and
0.6 mM, respectively. The rate of dehalogenation was
determined based on 0-, 5-, and 10-min data points.
View larger version (10K):
[in a new window]
Fig. 7.
Isotope inventory plot for the dehalogenation
of 3-chloro-4-hydroxybenzoate. Percent activity was plotted
versus percent D2O in the assay mixture. The
data were fitted to a linear regression equation.
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figures 1S and 2S.
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