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Reaction Mechanism and Stereochemistry of γ-Hexachlorocyclohexane Dehydrochlorinase LinA

2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês

10.1074/jbc.m007452200

1083-351X

Lukáš Trantı́rek, Kamila Hynková, Yuji Nagata, Alexey G. Murzin, Alena Ansorgová, Vladimı́r Sklenář, Jir̆ı́ Damborský,

Microbial Metabolic Engineering and Bioproduction

γ-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide γ-hexachlorocyclohexane (γ-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of γ-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 γ-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic γ-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 γ-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic γ-PCCH. δ-PCCH was detected as the only product of dehydrochlorination of δ-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 γ-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 γ-HCH to 1,2,4-TCB by LinA consists of two 1,2-anticonformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination. γ-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide γ-hexachlorocyclohexane (γ-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of γ-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 γ-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic γ-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 γ-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic γ-PCCH. δ-PCCH was detected as the only product of dehydrochlorination of δ-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 γ-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 γ-HCH to 1,2,4-TCB by LinA consists of two 1,2-anticonformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination. γ-hexachlorocyclohexane dehydrochlorinase from S. paucimobilis UT26 N-[1-(4-bromophenyl)ethyl]-5-fluoro-salicilamide gas chromatography-mass spectrometry hexachlorocyclohexane pentachlorocyclohexene nuclear magnetic resonance trichlorobenzene tetrachlorocyclohexa-1,4-diene density functional theory circular dichroism 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 (1Lipke H. Kearns C.W. J. Biol. Chem. 1959; 234: 2123-2132Abstract Full Text PDF PubMed Google Scholar) 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 (2Tanaka K. Kurihara N. Nakajima M. Pestic. Biochem. Physiol. 1976; 6: 392-399Crossref Scopus (38) Google Scholar). 3-Chloro-d-alanine dehydrochlorinase (3Nagasawa T. Ohkishi H. Kawasaki B. Yamano H. Hosono H. Tani Y. Yamada H. J. Biol. Chem. 1982; 257: 13749-13756Abstract Full Text PDF PubMed Google Scholar) from Pseudomonas putida employs the cofactor pyridoxal 5′-phosphate during catalysis. γ-Hexachlorocyclohexane dehydrochlorinase (LinA)1 (4Nagata Y. Hatta T. Imai R. Kimbara K. Fukuda M. Yano K. Takagi M. Biosci. Biotech. Biochem. 1993; 57: 1582-1583Crossref Scopus (55) Google Scholar) from the γ-hexachlorocyclohexane-degrading bacteriumSphingomonas paucimobilis UT26 catalyzes the conversion of γ-hexachlorocyclo hexane (γ-HCH) to 1,2,4-trichlorobenzene (1,2,4-TCB) via γ-1,3,4,5,6-pentachlorocyclohexene (γ-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 γ-hexachlorocyclohexane dehydrochlorinase was cloned by Imai et al. (5Imai R. Nagata Y. Fukuda M. Takagi M. Yano K. J. Bacteriol. 1991; 173: 6811-6819Crossref PubMed Google Scholar). 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. (6Thomas J.-C. Berger F. Jacquier M. Bernillon D. Baud-Grasset F. Truffaout N. Normand P. Vogel T.M. Simonet P. J. Bacteriol. 1996; 178: 6049-6055Crossref PubMed Google Scholar) from the newly isolated γ-HCH-degrading bacterium. The G+C content oflinA (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 (γ-HCH dehydrochlorinase LinA) was purified to homogeneity by Nagata et al. (4Nagata Y. Hatta T. Imai R. Kimbara K. Fukuda M. Yano K. Takagi M. Biosci. Biotech. Biochem. 1993; 57: 1582-1583Crossref Scopus (55) Google Scholar). Purified LinA showed activity with α-, γ-, and δ-HCH, but not with β-HCH. Because β-HCH does not contain a 1,2-biaxial HCl group, it was proposed that LinA dehydrochlorinates stereoselectively at this pair of hydrogen and chlorine (7Nagasawa S. Kikuchi R. Nagata Y. Takagi M. Matsuo M. Chemosphere. 1993; 26: 1187-1201Crossref Scopus (20) Google Scholar). This paper presents stereochemical analysis of the reaction products of enzymatic dehydrochlorination of γ-HCH by LinA. The absolute configuration and conformation of the reaction products is established, and the reaction mechanism of dehydrochlorination of LinA is proposed. γ-PCCH was synthesized by alkaline dehydrochlorination of γ-HCH (8Nakazima M. Okubo T. Katumura Y. Botyu-Kagaku. 1949; 14: 10-19Google Scholar). γ-HCH of more than 98% purity was purchased from Sigma-Aldrich. 50 mg of γ-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. γ-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. Purified enzyme LinA was prepared as described previously (4Nagata Y. Hatta T. Imai R. Kimbara K. Fukuda M. Yano K. Takagi M. Biosci. Biotech. Biochem. 1993; 57: 1582-1583Crossref Scopus (55) Google Scholar). 10 mg of γ-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 γ-PCCH was purified using the same procedure as described above for chemically synthesized γ-PCCH. Synthesized γ-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. 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. 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 spectra were collected on a Bruker AVANCE 500 MHz spectrometer equipped with a z-gradient triple resonance1H/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 (9Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3112) Google Scholar) with a mixing time of 50 ms and two-dimensional NOESY (10Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar) with a mixing time of 900 ms were acquired for resonance assignment of γ-PCCH. The acquisition parameters used for selective one-dimensional1H 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 (t 1,t 2), 2048 complex points in thet 2 dimension, 1024 complex points int 1, 2.2 s recycle delay and 32 scans. The spectrum was collected with the States-TPPI quadrature detection int 1 (11Marion D. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1989; 85: 393-399Google Scholar) with mixing time 900 ms. The spectrum was zero-filled to 2048 real points in t 2 and to 1024 real points in the t 1 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-dimensional1H 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. 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. 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. (12Kutzelnigg W. Fleischer U. Schindler M. NMR Basic Principles and Progress. Springer-Verlag, Berlin1990: 167-262Google Scholar) 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). The homology model of LinA dehalogenase was constructed using the method of satisfaction of spatial restraints as described elsewhere. 2Y. Nagata, K. Mori, M. Takagi, A. Murzin, and J. Damborsky, submitted for publication. The crystal structure of scytalone dehydratase (13Lundqvist T. Rice J. Hodge C.N. Basarab G.S. Pierce J. Lindqvist Y. Structure. 1994; 2: 937-944Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), nuclear transport factor-2 (14Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar), 3-oxo-Δ5-steroid isomerase (15Wu Z.R. Ebrahimian S. Zawrotny M.E. Thornburg L.D. Perez-Alvarado G.C. Brothers P. Pollack R.M. Summers M.F. Science. 1997; 276: 415-417Crossref PubMed Scopus (140) Google Scholar), and naphthalene 1,2-dioxygenase (16Kauppi B. Lee K. Carredano E. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. Structure. 1998; 6: 571-586Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) served as the template structures (PDB accession codes 1std, 1oun, 1opy, and 1ndo). The substrate molecule γ-HCH was docked in the active site manually using the program O, version 6.2.1. (17Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The numbering of the atoms in γ-HCH and γ-PCCH molecules used in the article is given in Fig. 1. The nomenclature of Izumi and Tai (18Izumi Y. Tai A. Stereo-differentiating Reactions: the Nature of Asymmetric Reactions. Kodansha, Tokyo1977Crossref Google Scholar) was used for classification of the stereochemical course of the reactions. The activity of LinA toward γ-PCCH, originating from the alkaline dehydrochlorination of γ-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 γ-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 γ-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 γ-PCCH by LinA. Chromatography of the γ-PCCH formed by enzymatic dehydrochlorination using chiral stationary phase confirmed the formation of a single enantiomer in the reaction mixture (Fig.2 A, inset). Alkaline dehydrochlorination of γ-HCH is known to proceed primarily by an E2(AxhDHDN according to IUPAC)anti elimination mechanism (19Cristol S.J. J. Am. Chem. Soc. 1946; 69: 338-342Crossref Scopus (39) Google Scholar, 20Cristol S.J. Hause N.L. Meek J.S. J. Am. Chem. Soc. 1950; 73: 674-679Crossref Scopus (43) Google Scholar) 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 γ-PCCH from alkaline dehydrochlorination is identical with the stereoisomer of γ-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. 2 B,inset). The formation of the single enantiomer during enzymatic dehydrochlorination of γ-HCH suggests that LinA enzyme specifically differentiates the enantiotopical pairs of the vicinal HCl.Figure 2GC-MS chromatograms of the reaction mixtures from dehydrochlorination of γ-HCH (A), synthetic γ-PCCH (B) and δ-HCH (C) by LinA enzyme. A single product (1,2,4-TCB) is produced by dehydrochlorination of γ-HCH, whereas two products (1,2,4-TCB and 1,2,3-TCB) are produced by dehydrochlorination of synthetic γ-PCCH. δ-HCH is dehydrochlorinated to δ-PCCH, which is not further transformed to TCB. The chromatograms presented in theinsets show separation of γ-PCCH on the chiral stationary phase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Different rates of formation of 1,2,4-TCB and 1,2,3-TCB were observed in kinetic measurements of dehydrochlorination of synthetic γ-PCCH by LinA enzyme (Fig. 3). At the same time, different rates of consumption of the two enantiomers of synthetic γ-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 γ-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 γ-PCCH enantiomers by LinA. The activity of LinA toward δ-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 γ-PCCH. The δ-PCCH was identified by GC-MS as the only product of the reaction. No activity of LinA toward δ-PCCH was observed (Fig.2 C). 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. Microwave spectroscopy and electron diffraction experiments showed the presence of only two possible conformations for the cyclohexene ring (21Scharpen L.H. Wollrab J.E. Ames D.P. J. Chem. Phys. 1968; 49: 2368-2372Crossref Scopus (32) Google Scholar, 22Chiang J.F. Bauer S.H. J. Am. Chem. Soc. 1969; 91: 1898-1901Crossref Scopus (139) Google Scholar). The conformational equilibrium of biosynthetic γ-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 α (Fig. 1 B) are3JH2-H3 = 2.5 Hz,3JH3-H4 = 8.3 Hz,3JH4-H5 = 2.2 Hz,3JH5-H6 = 2.4 Hz and for conformation β (Fig.1 C) 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 α conformation was found to be populated in the range of 82–92%. The population of the α conformation of 1,3(S),4(R),5(R),6(S)-PCCH was calculated to be 8–18%. The absolute configuration of the biosynthetic γ-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 γ-PCCH is a single enantiomer. (ii) The biosynthetic γ-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 (23Wiberg K., B. Stratmann R., E. Frisch M. Chem. Phys. Lett. 1998; 297: 60-64Crossref Scopus (249) Google Scholar,24Bour P. J. Phys. Chem. 1999; 103: 5099-5104Crossref Scopus (22) Google Scholar). Previous sequence searches for evolutionary relatives of LinA did not result in any significant hits (5Imai R. Nagata Y. Fukuda M. Takagi M. Yano K. J. Bacteriol. 1991; 173: 6811-6819Crossref PubMed Google Scholar). A PSI-BLAST (25Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) search for potential members of a new superfamily of proteins (26Murzin A. Curr. Opin. Struct. Biol. 1998; 8: 380-387Crossref PubMed Scopus (212) Google Scholar) revealed that LinA shows distant relationships with scytalone dehydratase (13Lundqvist T. Rice J. Hodge C.N. Basarab G.S. Pierce J. Lindqvist Y. Structure. 1994; 2: 937-944Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), nuclear transport factor-2 (14Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar), and 3-oxo-D5-steroid isomerase (15Wu Z.R. Ebrahimian S. Zawrotny M.E. Thornburg L.D. Perez-Alvarado G.C. Brothers P. Pollack R.M. Summers M.F. Science. 1997; 276: 415-417Crossref PubMed Scopus (140) Google Scholar).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 (26Murzin A. Curr. Opin. Struct. Biol. 1998; 8: 380-387Crossref PubMed Scopus (212) Google Scholar). 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 γ-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.6 B). 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 The following experimental observations have been taken into account for the proposed reaction mechanism of LinA. (i) γ-PCCH formed by the enzymatic dehydrochlorination of γ-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 γ-HCH, and (iv) His-73 and Asp-25 form the catalytic dyad of LinA. The putative reaction mechanism for dehydrochlorination of γ-HCH by LinA enzyme is depicted in Fig. 7. Molecular modeling revealed that the most probable conformation of the γ-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 (27Bartsch R. Zavada J. Chem. Rev. 1980; 6: 453-493Crossref Scopus (146) Google Scholar). 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 (13Lundqvist T. Rice J. Hodge C.N. Basarab G.S. Pierce J. Lindqvist Y. Structure. 1994; 2: 937-944Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 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 A dehydrochlorination of γ-PCCH is considered to proceed in two successive steps as shown in Reactions 1 and 2 (7Nagasawa S. Kikuchi R. Nagata Y. Takagi M. Matsuo M. Chemosphere. 1993; 26: 1187-1201Crossref Scopus (20) Google Scholar, 19Cristol S.J. J. Am. Chem. Soc. 1946; 69: 338-342Crossref Scopus (39) Google Scholar, 28Orloff H. Kolka A.J. J. Am. Chem. Soc. 1954; 76: 5484-5490Crossref Scopus (20) Google Scholar). C6H5Cl5→C6H4Cl4+HClEquation 1 C6H4Cl4→C6H3Cl3+HClEquation 2 We confirmed that LinA is specific toward 1,2-biaxialhydrogen and the chlorine pair in Reaction 1. Enzymatic dehydrochlorination of γ-PCCH proceeds by a 1,2-antidehydrochlorination 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 γ-HCH dehydrochlorination (Fig. 8). 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 (7Nagasawa S. Kikuchi R. Nagata Y. Takagi M. Matsuo M. Chemosphere. 1993; 26: 1187-1201Crossref Scopus (20) Google Scholar). 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 γ-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 (28Orloff H. Kolka A.J. J. Am. Chem. Soc. 1954; 76: 5484-5490Crossref Scopus (20) Google Scholar) 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. The chair conformation of γ-HCH is expected to be the active conformation during its dehydrochlorination by LinA (Fig.6 B) for two reasons. One is that γ-PCCH is known to be both a LinA substrate and a competitive inhibitor of γ-HCH dehydrochlorination (7Nagasawa S. Kikuchi R. Nagata Y. Takagi M. Matsuo M. Chemosphere. 1993; 26: 1187-1201Crossref Scopus (20) Google Scholar), and the chair conformation allows similar binding modes for both γ-HCH and γ-PCCH substrates. The other reason is that in a chair conformation there is at least oneaxial chlorine atom laying in the same plane as the abstracted proton. The fact that both γ-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 γ-HCH, the twist conformation of α is the expected active conformation for γ-PCCH. The production of 1,3(R),4(S),5(S),6(R)-PCCH during the enzymatic transformation of γ-HCH proves differentiation of the enantiotopic H2Cl3/H5Cl6 and H3Cl2/H6Cl5 groups. The different rates of the consumption of the two enantiomers of the γ-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 γ-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 γ-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. We thank Prof. Jaroslav Jonas for a critical reading of the manuscript and useful comments on its contents, Drs. Radek Marek and Jiřı́ Czernek for valuable discussions (Masaryk University, Brno, Czech Republic), Dr. Petr Bouř(Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic) for providing us with the program TABRN95 for visualization of the theoretical CD spectrum, Drs. Oldřich 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.