Reaction Mechanism and Stereochemistry of gamma -Hexachlorocyclohexane Dehydrochlorinase LinA*

Lukás TrantírekDagger , Kamila HynkováDagger , Yuji Nagata§, Alexey Murzin, Alena AnsorgováDagger , Vladimír SklenárDagger , and Jirí DamborskýDagger ||

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

gamma -Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma -hexachlorocyclohexane (gamma -HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of gamma -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 gamma -HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma -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 gamma -PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma -PCCH. delta -PCCH was detected as the only product of dehydrochlorination of delta -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 gamma -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 gamma -HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. gamma -Hexachlorocyclohexane dehydrochlorinase (LinA)1 (4) from the gamma -hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 catalyzes the conversion of gamma -hexachlorocyclo hexane (gamma -HCH) to 1,2,4-trichlorobenzene (1,2,4-TCB) via gamma -1,3,4,5,6-pentachlorocyclohexene (gamma -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 gamma -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 gamma -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 (gamma -HCH dehydrochlorinase LinA) was purified to homogeneity by Nagata et al. (4). Purified LinA showed activity with alpha -, gamma -, and delta -HCH, but not with beta -HCH. Because beta -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 gamma -HCH by LinA. The absolute configuration and conformation of the reaction products is established, and the reaction mechanism of dehydrochlorination of LinA is proposed.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemical Synthesis of gamma -PCCH-- gamma -PCCH was synthesized by alkaline dehydrochlorination of gamma -HCH (8). gamma -HCH of more than 98% purity was purchased from Sigma-Aldrich. 50 mg of gamma -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. gamma -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 gamma -PCCH-- Purified enzyme LinA was prepared as described previously (4). 10 mg of gamma -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 gamma -PCCH was purified using the same procedure as described above for chemically synthesized gamma -PCCH.

Kinetics of LinA with gamma -PCCH-- Synthesized gamma -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 gamma -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-Delta 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 gamma -HCH was docked in the active site manually using the program O, version 6.2.1. (17).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of the Reaction Products by GC-MS-- The numbering of the atoms in gamma -HCH and gamma -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 gamma -PCCH, originating from the alkaline dehydrochlorination of gamma -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 gamma -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 gamma -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 gamma -PCCH by LinA. Chromatography of the gamma -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 gamma -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 gamma -PCCH from alkaline dehydrochlorination is identical with the stereoisomer of gamma -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 gamma -HCH suggests that LinA enzyme specifically differentiates the enantiotopical pairs of the vicinal HCl.



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Fig. 1.   Numbering of atoms in gamma -HCH (A), gamma -PCCH conformer alpha  (B), and gamma -PCCH conformer beta  (C). Psi a, Psi 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 gamma -HCH (A), synthetic gamma -PCCH (B) and delta -HCH (C) by LinA enzyme. A single product (1,2,4-TCB) is produced by dehydrochlorination of gamma -HCH, whereas two products (1,2,4-TCB and 1,2,3-TCB) are produced by dehydrochlorination of synthetic gamma -PCCH. delta -HCH is dehydrochlorinated to delta -PCCH, which is not further transformed to TCB. The chromatograms presented in the insets show separation of gamma -PCCH on the chiral stationary phase.

Different rates of formation of 1,2,4-TCB and 1,2,3-TCB were observed in kinetic measurements of dehydrochlorination of synthetic gamma -PCCH by LinA enzyme (Fig. 3). At the same time, different rates of consumption of the two enantiomers of synthetic gamma -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 gamma -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 gamma -PCCH enantiomers by LinA. The activity of LinA toward delta -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 gamma -PCCH. The delta -PCCH was identified by GC-MS as the only product of the reaction. No activity of LinA toward delta -PCCH was observed (Fig. 2C).



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Fig. 3.   Kinetics of dehydrochlorination of synthetic gamma -PCCH by LinA. The stereoisomer 1,3(R),4(S),5(S),6(R)-PCCH (open circle , 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 (black-square, dashed line) is observed.

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 gamma -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.

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 gamma -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 alpha  (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 beta  (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 alpha  conformation was found to be populated in the range of 82-92%. The population of the alpha  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 gamma -PCCH-- The absolute configuration of the biosynthetic gamma -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 gamma -PCCH is a single enantiomer. (ii) The biosynthetic gamma -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).

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 gamma -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 gamma -hexachlorocyclohexane dehydrochlorinase LinA (B). Only the alpha -trace, catalytic dyads His-73/Asp-25 and the ligands are shown for clarity.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reaction Mechanism of gamma -HCH Dehydrochlorinase LinA-- The following experimental observations have been taken into account for the proposed reaction mechanism of LinA. (i) gamma -PCCH formed by the enzymatic dehydrochlorination of gamma -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 gamma -HCH, and (iv) His-73 and Asp-25 form the catalytic dyad of LinA.

The putative reaction mechanism for dehydrochlorination of gamma -HCH by LinA enzyme is depicted in Fig. 7. Molecular modeling revealed that the most probable conformation of the gamma -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 gamma -hexachlorocyclohexane dehydrochlorinase LinA. Step 1, nucleophilic attack of His-73 on gamma -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 gamma -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 gamma -HCH.

A dehydrochlorination of gamma -PCCH is considered to proceed in two successive steps as shown in Reactions 1 and 2 (7, 19, 28).
<UP>C<SUB>6</SUB>H<SUB>5</SUB>Cl<SUB>5</SUB></UP>→<UP>C<SUB>6</SUB>H<SUB>4</SUB>Cl<SUB>4</SUB></UP>+<UP>HCl</UP> (Eq. 1)

<UP>C<SUB>6</SUB>H<SUB>4</SUB>Cl<SUB>4</SUB></UP>→<UP>C<SUB>6</SUB>H<SUB>3</SUB>Cl<SUB>3</SUB></UP>+<UP>HCl</UP> (Eq. 2)
We confirmed that LinA is specific toward 1,2-biaxial hydrogen and the chlorine pair in Reaction 1. Enzymatic dehydrochlorination of gamma -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 gamma -HCH dehydrochlorination (Fig. 8).



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Fig. 8.   Pathway scheme for the enzymatic dehydrochlorination of gamma -HCH (A) and racemic gamma -PCCH (B) by LinA. Dehydrochlorination of biosynthetic gamma -PCCH results in formation of 1,2,4-TCB, whereas the dehydrochlorination of synthetic gamma -PCCH gives rise to 1,2,4-TCB and 1,2,3-TCB. Step 1, gamma -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.

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 gamma -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 gamma -HCH Dehydrochlorinase Substrates-- The chair conformation of gamma -HCH is expected to be the active conformation during its dehydrochlorination by LinA (Fig. 6B) for two reasons. One is that gamma -PCCH is known to be both a LinA substrate and a competitive inhibitor of gamma -HCH dehydrochlorination (7), and the chair conformation allows similar binding modes for both gamma -HCH and gamma -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 gamma -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 gamma -HCH, the twist conformation of alpha  is the expected active conformation for gamma -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 gamma -HCH proves differentiation of the enantiotopic H2Cl3/H5Cl6 and H3Cl2/H6Cl5 groups. The different rates of the consumption of the two enantiomers of the gamma -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 gamma -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 gamma -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 Jirí Czernek for valuable discussions (Masaryk University, Brno, Czech Republic), Dr. Petr Bour (Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic) for providing us with the program TABRN95 for visualization of the theoretical CD spectrum, Drs. Oldrich 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, gamma -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Lipke, H., and Kearns, C. W. (1959) J. Biol. Chem. 234, 2123-2132[Free Full Text]
2. Tanaka, K., Kurihara, N., and Nakajima, M. (1976) Pestic. Biochem. Physiol. 6, 392-399
3. Nagasawa, T., Ohkishi, H., Kawasaki, B., Yamano, H., Hosono, H., Tani, Y., and Yamada, H. (1982) J. Biol. Chem. 257, 13749-13756[Abstract/Free Full Text]
4. Nagata, Y., Hatta, T., Imai, R., Kimbara, K., Fukuda, M., Yano, K., and Takagi, M. (1993) Biosci. Biotech. Biochem. 57, 1582-1583
5. Imai, R., Nagata, Y., Fukuda, M., Takagi, M., and Yano, K. (1991) J. Bacteriol. 173, 6811-6819[Medline] [Order article via Infotrieve]
6. Thomas, J.-C., Berger, F., Jacquier, M., Bernillon, D., Baud-Grasset, F., Truffaout, N., Normand, P., Vogel, T. M., and Simonet, P. (1996) J. Bacteriol. 178, 6049-6055[Abstract]
7. Nagasawa, S., Kikuchi, R., Nagata, Y., Takagi, M., and Matsuo, M. (1993) Chemosphere 26, 1187-1201
8. Nakazima, M., Okubo, T., and Katumura, Y. (1949) Botyu-Kagaku 14, 10-19
9. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528
10. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553[CrossRef]
11. Marion, D., Ikura, M., Tschudin, R., and Bax, A. (1989) J. Magn. Reson. 85, 393-399
12. Kutzelnigg, W., Fleischer, U., and Schindler, M. (1990) NMR Basic Principles and Progress , pp. 167-262, Springer-Verlag, Berlin
13. Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J., and Lindqvist, Y. (1994) Structure 2, 937-944[Medline] [Order article via Infotrieve]
14. Bullock, T. L., Clarkson, W. D., Kent, H. M., and Stewart, M. (1996) J. Mol. Biol. 260, 422-431[CrossRef][Medline] [Order article via Infotrieve]
15. Wu, Z. R., Ebrahimian, S., Zawrotny, M. E., Thornburg, L. D., Perez-Alvarado, G. C., Brothers, P., Pollack, R. M., and Summers, M. F. (1997) Science 276, 415-417[Abstract/Free Full Text]
16. Kauppi, B., Lee, K., Carredano, E., Parales, R. E., Gibson, D. T., Eklund, H., and Ramaswamy, S. (1998) Structure 6, 571-586[Medline] [Order article via Infotrieve]
17. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
18. Izumi, Y., and Tai, A. (1977) Stereo-differentiating Reactions: the Nature of Asymmetric Reactions , Kodansha, Tokyo
19. Cristol, S. J. (1946) J. Am. Chem. Soc. 69, 338-342
20. Cristol, S. J., Hause, N. L., and Meek, J. S. (1950) J. Am. Chem. Soc. 73, 674-679
21. Scharpen, L. H., Wollrab, J. E., and Ames, D. P. (1968) J. Chem. Phys. 49, 2368-2372
22. Chiang, J. F., and Bauer, S. H. (1969) J. Am. Chem. Soc. 91, 1898-1901
23. Wiberg, K., B., Stratmann, R., E., and Frisch, M. (1998) Chem. Phys. Lett. 297, 60-64[CrossRef]
24. Bour, P. (1999) J. Phys. Chem. 103, 5099-5104
25. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
26. Murzin, A. (1998) Curr. Opin. Struct. Biol. 8, 380-387[CrossRef][Medline] [Order article via Infotrieve]
27. Bartsch, R., and Zavada, J. (1980) Chem. Rev. 6, 453-493
28. Orloff, H., and Kolka, A. J. (1954) J. Am. Chem. Soc. 76, 5484-5490


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