Reaction Mechanism and Stereochemistry of
-Hexachlorocyclohexane Dehydrochlorinase LinA*
Luká
Trantírek
,
Kamila
Hynková
,
Yuji
Nagata§,
Alexey
Murzin¶,
Alena
Ansorgová
,
Vladimír
Sklená
, and
Ji
í
Damborský
From the
Laboratory of Biomolecular Structure and
Dynamics, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic,
the § Institute of Genetic Ecology, Tohoku University,
Katahira, Sendai 980-8577, Japan, and the ¶ Centre for Protein
Engineering, MRC Centre, Hills Road, Cambridge, CB2 2QH, United
Kingdom
Received for publication, August 16, 2000, and in revised form, November 13, 2000
 |
ABSTRACT |
-Hexachlorocyclohexane dehydrochlorinase
(LinA) catalyzes the initial steps in the biotransformation of
the important insecticide
-hexachlorocyclohexane (
-HCH) by the
soil bacterium Sphingomonas paucimobilis UT26.
Stereochemical analysis of the reaction products formed during
conversion of
-HCH by LinA was investigated by GC-MS, NMR, CD, and
molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from
-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic
-PCCH were converted by LinA, each at a different rate.
1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of
the biotransformation of biosynthetic
-PCCH. 1,2,4-TCB and 1,2,3-TCB
were identified as the dehydrochlorination products of racemic
-PCCH.
-PCCH was detected as the only product of
dehydrochlorination of
-HCH. LinA requires the presence of a
1,2-biaxial HCl pair on a substrate molecule. LinA
enantiotopologically differentiates two 1,2-biaxial HCl
pairs present on
-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH.
Furthermore, LinA enantiomerically differentiates
1,3(S),4(R),5(R),6(S)-PCCH
and
1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of
-HCH to 1,2,4-TCB by LinA consists of two 1,2-anti
conformationally dependent dehydrochlorinations followed by
1,4-anti dehydrochlorination.
 |
INTRODUCTION |
Dehydrochlorinases are enzymes that eliminate HCl from a substrate
molecule leading to the formation of a double bond. Three different
dehydrochlorinases have been reported to date. DDT dehydrochlorinase (1) isolated from Musca domestica catalyzes
dehydrochlorination of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane to
1,1-dichloro-2,2-bis(4-chlorophenyl) ethene and requires glutathione
for its activity (2). 3-Chloro-D-alanine dehydrochlorinase
(3) from Pseudomonas putida employs the cofactor pyridoxal
5'-phosphate during catalysis.
-Hexachlorocyclohexane dehydrochlorinase (LinA)1 (4)
from the
-hexachlorocyclohexane-degrading bacterium
Sphingomonas paucimobilis UT26 catalyzes the conversion of
-hexachlorocyclo hexane (
-HCH) to 1,2,4-trichlorobenzene
(1,2,4-TCB) via
-1,3,4,5,6-pentachlorocyclohexene (
-PCCH).
LinA does not require any cofactor for its activity and therefore
represents a distinct type of enzyme from the former two dehydrochlorinases.
The linA gene encoding
-hexachlorocyclohexane
dehydrochlorinase was cloned by Imai et al. (5). The
nucleotide sequence of the linA did not show sequence
similarity to any sequence deposited in the databases. Recently, a gene
identical to linA was cloned by Thomas et al. (6)
from the newly isolated
-HCH-degrading bacterium. The G+C content of
linA (53%) is considerably lower than that of other genes
and of the total DNA of Sp. paucimobilis strains, suggesting
that linA originates from the genome of some other genus or
organism. The linA gene was expressed in Escherichia coli, and the translation product (
-HCH dehydrochlorinase LinA) was purified to homogeneity by Nagata et al. (4). Purified LinA showed activity with
-,
-, and
-HCH, but not with
-HCH. Because
-HCH does not contain a 1,2-biaxial HCl
group, it was proposed that LinA dehydrochlorinates stereoselectively
at this pair of hydrogen and chlorine (7).
This paper presents stereochemical analysis of the reaction products of
enzymatic dehydrochlorination of
-HCH by LinA. The absolute
configuration and conformation of the reaction products is established,
and the reaction mechanism of dehydrochlorination of LinA is proposed.
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EXPERIMENTAL PROCEDURES |
Chemical Synthesis of
-PCCH--
-PCCH was synthesized by
alkaline dehydrochlorination of
-HCH (8).
-HCH of more than 98%
purity was purchased from Sigma-Aldrich. 50 mg of
-HCH was dissolved
in 5 ml of acetonitrile. The synthesis was started by addition of 2.5 ml of 0.1 M NaOH to the reaction mixture. The reaction
mixture was heated for 20 min at 40 °C. The products of synthesis
were extracted with hexane.
-PCCH was purified by preparative liquid
chromatography with a steel column (8 × 250 mm) packed by silica
gel (7 µm). 20% dichloromethane in hexane was used as the mobile phase.
Biochemical Synthesis of
-PCCH--
Purified enzyme LinA was
prepared as described previously (4). 10 mg of
-HCH and 100 ml of
phosphate buffer, pH 7.5, were equilibrated at 35 °C in a shaking
water bath. The enzymatic reaction was initiated by adding 100 µl of
LinA (protein concentration: 45 mg/l). The reaction was stopped after 5 min by extraction with hexane. The product
-PCCH was purified using
the same procedure as described above for chemically synthesized
-PCCH.
Kinetics of LinA with
-PCCH--
Synthesized
-PCCH was
dissolved in phosphate buffer (10 ml, pH 7.5) and equilibrated at
35 °C in a shaking water bath. The enzymatic reaction was initiated
by adding 10 µl of LinA (protein concentration: 45 mg/l). The
progress of the reaction was monitored in 1 ml of the reaction mixture
at 5, 10, 20, 40 min, and 24 h. Samples from the reaction mixture
were extracted with 0.3 ml of hexane and analyzed by GC-MS as described below.
GC-MS Analysis--
Reaction products were identified and
quantified on GC-MS system (Hewlett Packard 6890) with helium as a
carrier gas. The temperature of the DB-5MS capillary column (59.5 m × 0.25 mm × 0.25 µm, J&W Scientific) was kept at
50 °C for 2 min and then increased to 300 °C at a rate of
15 °C/min. The scan mode at 50-550 m/z was
used for searching and for identification of products, whereas SIM mode
was used for quantification.
Testing of Enantiomerical Purity--
The enantiomerical purity
of the reaction products was monitored on a GC system (Hewlett Packard
5890) equipped with ECD detector and CYCLODEX-B capillary column (30 m × 0.25 mm × 0.25 µm, J&W Scientific). The column
temperature was increased from 80 to 220 °C at a rate 10 °C/min
and then the temperature was kept for 10 min at 220 °C.
NMR Spectroscopy--
NMR spectra were collected on a Bruker
AVANCE 500 MHz spectrometer equipped with a z-gradient triple resonance
1H/13C/BB probehead at 298.2 K. The NMR samples
were prepared in total volumes of 260 µl in 99.99%
CD3CN. Selective one-dimensional 1H TOCSY (9)
with a mixing time of 50 ms and two-dimensional NOESY (10) with a
mixing time of 900 ms were acquired for resonance assignment of
-PCCH. The acquisition parameters used for selective one-dimensional
1H TOCSY were: spectral width 5000 Hz, 8192 complex points,
2.2 s recycle delay, mixing time 50 ms and 8 scans. The spectrum
was zero-filled to 12288 real points, and resolution was enhanced by
82o shifted-square sine bell apodization function. The
acquisition parameters used for two-dimensional NOESY were: spectral
width 5000 Hz in the both dimensions (t1,
t2), 2048 complex points in the
t2 dimension, 1024 complex points in
t1, 2.2 s recycle delay and 32 scans. The
spectrum was collected with the States-TPPI quadrature detection in
t1 (11) with mixing time 900 ms. The spectrum
was zero-filled to 2048 real points in t2 and to
1024 real points in the t1 dimension, and
resolution was enhanced by a 82o shifted-square sine bell
apodization function. Three bond proton-proton scalar coupling
constants were obtained from standard high resoluted one-dimensional
1H NMR spectrum. The acquisition parameters were: spectral
width 5000 Hz, 12288 complex points, 2.2 s recycle delay, and 16 scans. The spectrum was zero-filled to 32 120 real points, and the
resolution was enhanced using a 45o shifted-square sine
bell apodization function.
CD Spectroscopy--
The CD spectra were measured in
acetonitrile at 298.2 K using a Jasco J-720 spectropolarimeter using a
1-cm path length and a wavelength of 200-350 nm.
Quantum Chemical Calculations--
Ab initio geometry
optimizations were conducted with Gaussian 98 (Gaussian, Inc.) using
density functional theory (DFT) method. These optimizations employed
the Becke3P86 hybrid functional and 6-31G** basis set. The scalar
couplings were calculated using the program deMon-NMR (MASTERS-CS,
Universite de Montreal, Canada). The PERDEW functional and the basis
set IGLO-III of Kutzelnigg et al. (12) were used in the
calculations. The electric and magnetic transition moments,
respectively, were calculated for the six energetically lowest
transitions using the time-dependent adiabatic extension of
DFT, Becke3P86 hybrid functional and 6-31++G** basis set.
Quantum-mechanical calculations were performed on a SGI R10000 (SGI).
Molecular Modeling--
The homology model of LinA dehalogenase
was constructed using the method of satisfaction of spatial restraints
as described elsewhere.2 The
crystal structure of scytalone dehydratase (13), nuclear transport
factor-2 (14), 3-oxo-
5-steroid isomerase (15), and
naphthalene 1,2-dioxygenase (16) served as the template structures (PDB
accession codes 1std, 1oun, 1opy, and 1ndo). The substrate molecule
-HCH was docked in the active site manually using the program O,
version 6.2.1. (17).
 |
RESULTS |
Identification of the Reaction Products by GC-MS--
The
numbering of the atoms in
-HCH and
-PCCH molecules used in the
article is given in Fig. 1. The
nomenclature of Izumi and Tai (18) was used for classification of the
stereochemical course of the reactions. The activity of LinA toward
-PCCH, originating from the alkaline dehydrochlorination of
-HCH,
was tested, and the end products of the reaction were identified using
GC-MS. These products were compared with the end products of the
enzymatic transformation of
-HCH (Fig.
2, A and B). The
same compound, 1,2,4-trichlorobenzene (1,2,4-TCB), was identified as
the product of dehydrochlorination of both synthetic and biosynthetic
-PCCH. In addition to 1,2,4-TCB, 1,2,3-TCB was also found in the
reaction mixture obtained from the dehydrochlorination of synthetic
-PCCH by LinA. Chromatography of the
-PCCH formed by enzymatic
dehydrochlorination using chiral stationary phase confirmed the
formation of a single enantiomer in the reaction mixture (Fig.
2A, inset). Alkaline dehydrochlorination
of
-HCH is known to proceed primarily by an E2
(AxhDHDN according to IUPAC)
anti elimination mechanism (19, 20) resulting in formation
of the racemate of
1,3(S),4(R),5(R),6(S)- and
1,3(R),4(S),5(S),6(R)-PCCH.
Consequently, one of the stereoisomers present in the racemate of
-PCCH from alkaline dehydrochlorination is identical with the
stereoisomer of
-PCCH formed during enzymatic dehydrochlorination,
whereas 1,2,3-TCB is the product formed by dehydrochlorination of the
remaining enantiomer present in the racemate (Fig. 2B,
inset). The formation of the single enantiomer during
enzymatic dehydrochlorination of
-HCH suggests that LinA enzyme
specifically differentiates the enantiotopical pairs of the vicinal
HCl.

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Fig. 1.
Numbering of atoms in
-HCH (A),
-PCCH conformer (B), and -PCCH
conformer (C).
a, b, a, and e
denote pseudo-axial, pseudo-equatorial, axial, and equatorial
positions, respectively.
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Fig. 2.
GC-MS chromatograms of the reaction mixtures
from dehydrochlorination of -HCH
(A), synthetic -PCCH
(B) and -HCH
(C) by LinA enzyme. A single product (1,2,4-TCB)
is produced by dehydrochlorination of -HCH, whereas two products
(1,2,4-TCB and 1,2,3-TCB) are produced by dehydrochlorination of
synthetic -PCCH. -HCH is dehydrochlorinated to -PCCH, which is
not further transformed to TCB. The chromatograms presented in the
insets show separation of -PCCH on the chiral stationary
phase.
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Different rates of formation of 1,2,4-TCB and 1,2,3-TCB were observed
in kinetic measurements of dehydrochlorination of synthetic
-PCCH by
LinA enzyme (Fig. 3). At the same time,
different rates of consumption of the two enantiomers of synthetic
-PCCH were observed by gas chromatographic analysis employing the
column for chiral separations. The enzyme eventually transformed all compounds present in the racemate. The different rates of the consumption of the two enantiomers of the
-PCCH as well as different rates of the creation of the 1,2,3- and 1,2,4-TCB suggests the enantiomerical differentiation of the
-PCCH enantiomers by LinA. The
activity of LinA toward
-HCH was tested, and the end products of the
reaction were identified to confirm specificity of LinA toward the
1,2-biaxial HCl pair during enzymatic dehydrochlorination of
-PCCH. The
-PCCH was identified by GC-MS as the only product of
the reaction. No activity of LinA toward
-PCCH was observed (Fig.
2C).

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Fig. 3.
Kinetics of dehydrochlorination of
synthetic -PCCH by LinA. The stereoisomer
1,3(R),4(S),5(S),6(R)-PCCH
( , solid line) is depleted at a different rate than the
stereoisomer
1,3(S),4(R),5(R),6(S)-PCCH
( , solid line). Corresponding formation of the products 1,2,4-TCB
( , dashed line) and 1,2,3-TCB ( , dashed
line) is observed.
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Stereochemical Analysis of the Reaction Products by NMR--
NMR
spectroscopy was used for the analysis of configuration and
conformation of synthetic and biosynthetic PCCH. The NMR spectra of
synthetic and biosynthetic PCCH were compared and found to be
identical. This result confirmed that alkaline dehydrochlorination gives rise to a product with the same relative configuration as enzymatic dehydrochlorination. This observation is in agreement with
GC-MS experiments. The configuration of the biosynthetic PCCH was
independently established by quantitative analysis of the experimental
three bond proton-proton scalar coupling constants, intensities, and
line widths of the appropriate resonances. The NMR spectrum (Fig.
4) corresponds to the enantiomorphic pair
1,3(R),4(S),5 (S),6(R)-PCCH/1,3(S),4(R),5(R),6(S)-PCCH,
which is the only possible product of anti elimination.

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Fig. 4.
One-dimensional 1H NMR spectrum
of biosynthetic -PCCH. Complete
(A) and magnified parts (B and C) of
the spectrum are presented. The signal at 6.13 ppm was assigned to the
olefinic proton 2, and the signal at 4.8 ppm was assigned to
the pseudoaxial allylic proton 3 (see Fig. 1). The protons 5 and 6 were
assigned to the signals a (4.7 pmm) and b (4.69 ppm), respectively. These protons form either
equatorial-pseudoequatorial/pseudoaxial or axial-pseudoequatorial
interproton pair. The signal c was assigned to the axial
proton 4.
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Microwave spectroscopy and electron diffraction experiments showed the
presence of only two possible conformations for the cyclohexene ring
(21, 22). The conformational equilibrium of biosynthetic
-PCCH (in
CD3CN at 298.2 K) was determined by fitting the weighted
averages of selected theoretical scalar couplings to the experimental
data. The theoretical three bond interproton scalar coupling constants
of
1,3(R),4(S),5(S),6(R)-PCCH
for conformation
(Fig. 1B) are
3JH2-H3 = 2.5 Hz,
3JH3-H4 = 8.3 Hz,
3JH4-H5 = 2.2 Hz,
3JH5-H6 = 2.4 Hz and for conformation
(Fig.
1C) are 5.8 Hz, 2.4 Hz, 2.0 Hz, and 5.9 Hz. Experimentally
derived scalar couplings were: 3JH2-H3 = 3.1 Hz, 3JH3-H4 = 7.8 Hz,
3JH4-H5 < 3 Hz,
3JH5-H6 < 3 Hz. For
1,3(R),4(S),5(S),6(R)-PCCH,
the
conformation was found to be populated in the range of
82-92%. The population of the
conformation of
1,3(S),4(R),5(R),6(S)-PCCH
was calculated to be 8-18%.
Determination of the Absolute Configuration of the Biosynthetic
-PCCH--
The absolute configuration of the biosynthetic
-PCCH
was determined by comparison of experimental and theoretical CD
spectra. The following assumptions were made prior to calculating the
CD spectrum. (i) The biosynthetic
-PCCH is a single enantiomer. (ii)
The biosynthetic
-PCCH is either
1,3(R),4(S),5(S),6(R)-PCCH or its enantiomer
1,3(S),4(R),5(R),6(S)-PCCH,
and (iii) the major conformation is ~87%. The theoretical and
experimental CD spectra are compared on Fig.
5. The experimental CD spectrum
corresponds well to the theoretical CD spectrum for
1,3(R),4(S),5(S),6(R)-PCCH enantiomer. The small difference in the excitation energy between the
experimental and theoretical spectra (about 20 nm) is an artifact of
DFT calculation and has been described previously by other authors (23,
24).

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Fig. 5.
Scaled theoretical CD spectrum of the
1,3(R),4(S),5(S),6(R)-PCCH
(dotted line), the scaled theoretical CD spectrum of
the
1,3(S),4(R),5(R),6(S)-PCCH
(dashed line), and experimental CD spectrum of
biosynthetic PCCH (solid line).
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Construction of the Theoretical Model of the Enzyme-Substrate
Complex--
Previous sequence searches for evolutionary relatives of
LinA did not result in any significant hits (5). A PSI-BLAST (25) search for potential members of a new superfamily of proteins (26)
revealed that LinA shows distant relationships with scytalone dehydratase (13), nuclear transport factor-2 (14), and
3-oxo-D5-steroid isomerase (15).2 The proteins
in this superfamily have diverged beyond notable sequence similarity
and have evolved different function, but retain the general design of
the active site cavity (26). This enabled us to construct a
three-dimensional model of LinA by homology and dock the substrate
molecule in its active site. A molecule of
-HCH was manually docked
into the LinA active site in a way that would allow efficient
abstraction of a hydrogen from the 1,2-biaxial HCl pair of
the substrate molecule by the general base His-73 (Fig.
6B). The theoretical model of
LinA complexed with the substrate is compared with the crystal
structure of scytalone dehydratase complexed with its inhibitor (Fig.
6). The figure illustrates the common fold and conserved catalytic dyad
His-73/Asp-25 of these enzymes. The essential role of the putative
catalytic dyad for LinA activity was confirmed experimentally by
site-directed mutagenesis.2

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Fig. 6.
Comparison of the crystal structure of
scytalone dehydratase (13) (A) and the theoretical
model of -hexachlorocyclohexane
dehydrochlorinase LinA (B). Only the -trace,
catalytic dyads His-73/Asp-25 and the ligands are shown for
clarity.
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 |
DISCUSSION |
Reaction Mechanism of
-HCH Dehydrochlorinase LinA--
The
following experimental observations have been taken into account for
the proposed reaction mechanism of LinA. (i)
-PCCH formed by the
enzymatic dehydrochlorination of
-HCH is in the configuration
corresponding to anti elimination. (ii) LinA exclusively dehydrochlorinates HCH substrates containing at least one
1,2-biaxial pair of hydrogen and chlorine. (iii)
1,3(R),4(S),5(S),6(R)-PCCH is the exclusive product of enzymatic dehydrochlorination of
-HCH, and (iv) His-73 and Asp-25 form the catalytic dyad of LinA.
The putative reaction mechanism for dehydrochlorination of
-HCH by
LinA enzyme is depicted in Fig. 7.
Molecular modeling revealed that the most probable conformation of the
-HCH in the active site is the chair conformation (see next
paragraph). The HCl pair involved in the reaction is forced to adopt
the 1,2-biaxial position in the enzyme active site. The
requirement for the presence of a 1,2-biaxial HCl pair in
the substrate molecules, the geometry of the active site as well as the
kinetic measurements, indicate an E2-like
dehydrochlorination mechanism (27). We propose that His-73 acts as the
base and attacks the hydrogen atom on C3, resulting in breaking of the
C3-H bond based on the analogous role of catalytic histidine in the
scytalone dehydratase (13). Asp-25 assists in the catalysis by keeping
His-73 in the proper orientation and by stabilizing the positive charge
that develops on the histidine imidazole ring during the reaction.
There are probably other residues, which stabilize the transition state
and the reaction products, e.g. via nonbonding interactions
with hydrogen and chlorine atoms on the ring. Possible candidates are
Lys-20 and Arg-129, which were shown to be important for the catalytic
activity of LinA by site-directed mutagenesis
experiments.2

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Fig. 7.
Putative reaction mechanism of
-hexachlorocyclohexane dehydrochlorinase LinA.
Step 1, nucleophilic attack of His-73 on -HCH, proton
abstraction, Cl formation, and its release. Step
2, release of
1,3(R),4(S),5(S),6(R)-PCCH,
release of H+, and repeated binding of
1,3(R),4(S),5(S),6(R)-PCCH
(alternatively,
1,3(R),4(S),5(S),6(R)-PCCH
turns around in the active site without leaving it). Step 3,
nucleophilic attack of His-73 on -PCCH, proton abstraction,
Cl formation, and its release. Step 4, release
of 1,3(R),4,6(R)-TDCN, release of H+,
and binding of -HCH.
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A dehydrochlorination of
-PCCH is considered to proceed in two
successive steps as shown in Reactions 1 and 2 (7, 19, 28).
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(Eq. 1)
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(Eq. 2)
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We confirmed that LinA is specific toward 1,2-biaxial
hydrogen and the chlorine pair in Reaction 1. Enzymatic
dehydrochlorination of
-PCCH proceeds by a 1,2-anti
dehydrochlorination reaction (Reaction 1), followed by
1,4-anti dehydrochlorination (Reaction 2). Because the
enzymatic transformation of
1,3(R),4(S),5(S),6(R)-PCCH results exclusively in the formation of 1,2,4-TCB, the reaction must
proceed through
1,3(R),4,6(R)-tetrachlorocyclohexa-1,4-diene (TCDN) as an intermediate (Fig. 8).
Biotransformation of
1,3(S),4(R),5(R),6(S)-PCCH to 1,2,3-TCB then proceeds through 1,3,5,6-TCDN. The
dehydrochlorination of the
1,3(R),4(S),5(S),6(R)-PCCH
by LinA starts on the H4Cl5 pair and proceeds analogously from a
stereochemical and mechanistic point of view to
-HCH
dehydrochlorination (Fig. 8).

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Fig. 8.
Pathway scheme for the enzymatic
dehydrochlorination of -HCH
(A) and racemic -PCCH
(B) by LinA. Dehydrochlorination of biosynthetic
-PCCH results in formation of 1,2,4-TCB, whereas the
dehydrochlorination of synthetic -PCCH gives rise to 1,2,4-TCB and
1,2,3-TCB. Step 1, -HCH; step 2,
1,3(R),4(S),5(S),6(R)-PCCH;
step 3, 1,3(R),4,6(R)-TCDN; step
4, 1,2,4-TCB; step 5,
1,3(S),4(R),5(R),6(S)-PCCH;
step 6, 1,3,5,6-TCDN; step 7, 1,2,3-TCB.
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Transformation of 1,3(R),4,6(R)-TCDN to 1,2,4-TCB
has been proposed to proceed by a spontaneous nonenzymatic
rearrangement, based on the assumption of an unstable diene-type
structure (7). 1,3(R),4,6(R)-TCDN has never been
directly detected in the reaction mixture, suggesting that 1,4 elimination of HCl from TCDN proceeds by the same or higher rate than
enzymatic 1,2 elimination of HCl from
-PCCH. 1,2,3-TCB is the
exclusive product of a 1,4 elimination reaction of 1,3,5,6-TCDN. The
specific formation of 1,2,3-TCB seems to support the enzymatic nature
of Reaction 2. LinA could specifically differentiate between 1,4 H6Cl3
and H3Cl6 groups of 1,3,5,6-TCDN, when only elimination of H6Cl3
results in formation of 1,2,3-TCB. In case of nonenzymatic elimination,
the preference for 1,4 elimination of H6Cl3 over H3Cl6, and lack of
1,3,5-TCB product (28) could be caused by the higher activation barrier or unfavorable thermodynamics of H3Cl6 elimination. More research is needed to elucidate the mechanism of Reaction 2.
Active Conformations of
-HCH Dehydrochlorinase
Substrates--
The chair conformation of
-HCH is expected to be
the active conformation during its dehydrochlorination by LinA (Fig.
6B) for two reasons. One is that
-PCCH is known to be
both a LinA substrate and a competitive inhibitor of
-HCH
dehydrochlorination (7), and the chair conformation allows similar
binding modes for both
-HCH and
-PCCH substrates. The other
reason is that in a chair conformation there is at least one
axial chlorine atom laying in the same plane as the
abstracted proton. The fact that both
-PCCH enantiomers are LinA
substrates strongly suggests that there may be more than one
substrate-binding mode. Based on the shape of the active site and
conformational analogy with the active conformation of
-HCH, the
twist conformation of
is the expected active conformation for
-PCCH.
Stereodifferentiation of the Substrates Dehydrochlorinated by
LinA--
The production of
1,3(R),4(S),5(S),6(R)-PCCH
during the enzymatic transformation of
-HCH proves differentiation
of the enantiotopic H2Cl3/H5Cl6 and H3Cl2/H6Cl5 groups. The different
rates of the consumption of the two enantiomers of the
-PCCH as well
as different rates of the creation of the 1,2,3- and 1,2,4-TCB confirm
the enantiomerical differentiation of the
-PCCH enantiomers by LinA. The production of the 1,2,4-TCB from
1,3(R),4(S),5(S),6(R)-PCCH by enzymatic dehydrochlorination proves differentiation of the diastereotopical H4Cl5 and H5Cl4 groups. Both topological
differentiations are consequences of sharing a common conformation on
one side of the ring facing the catalytic residues, the enantiomerical differentiation of
-PCCH enantiomers arises as a consequence of the
opposite orientation of the hydrogen and chlorine atoms on the double
bond at the active site. Whereas catalytic residues cause the
topological differentiation, the enantiomerical differentiation is
driven by the noncovalent interaction of the double bond substituents with noncatalytic residues in the active site of the LinA. The expected
active conformations of
1,3(R),4(S),5(S),6(R)-PCCH
and 1,3(S),4(R),5(R),6(S)-PCCH
are depicted in Fig. 8.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Jaroslav Jonas for a critical
reading of the manuscript and useful comments on its contents, Drs.
Radek Marek and Ji
í Czernek for valuable discussions
(Masaryk University, Brno, Czech Republic), Dr. Petr Bou
(Institute of Organic Chemistry and Biochemistry, Prague, Czech
Republic) for providing us with the program TABRN95 for visualization
of the theoretical CD spectrum, Drs. Old
ich Vrána and
Hanka Loskotová (Institute of Biophysics, Brno, Czech Republic)
for assistance with measurement of the CD spectra. Prof. Juli Feigon
(University of California, Los Angeles, CA) is acknowledged for help
with the linguistic revision of the manuscript.
 |
FOOTNOTES |
*
This project was supported by Grant Postdoc 203/97/P149 from
the Czech Grant Agency and by Grants ME276/1998 and VS96095 from the
Czech Ministry of Education.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. Fax:
420-5-41129506; E-mail: jiri@chemi.muni.cz.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M007452200
2
Y. Nagata, K. Mori, M. Takagi, A. Murzin, and J. Damborsky, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
LinA,
-hexachlorocyclohexane dehydrochlorinase from S. paucimobilis UT26;
BFS, N-[1-(4-bromophenyl)ethyl]-5-fluoro-salicilamide;
GC-MS, gas chromatography-mass spectrometry;
HCH, hexachlorocyclohexane;
PCCH, pentachlorocyclohexene;
NMR, nuclear
magnetic resonance;
TCB, trichlorobenzene;
TCDN, tetrachlorocyclohexa-1,4-diene;
DFT, density functional theory;
CD, circular dichroism.
 |
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