New metabolites in the degradation of α- and γ- hexachlorocyclohexane (HCH): Pentachlorocyclohexenes are hydroxylated to cyclohexenols and cyclohexenediols by the haloalkane dehalogenase LinB from Sphingobium indicum B90A

Article abstract of DOI:10.1021/jf800465q

Technical hexachlorocyclohexane (HCH) and lindane are obsolete pesticides whose former production and use led to widespread contaminations posing serious and lasting health and environmental risks. Out of nine possible stereoisomers, α-, β-, γ-, and -HCH are usually present at contaminated sites, and research for a better understanding of their biodegradation has become essential for the development of appropriate remediation technologies. Because haloalkane dehalogenase LinB was recently found responsible for the hydroxylation of β-HCH, δ-HCH, and δ-pentachlorocyclohexene (δ-PCCH), we decided to examine whether β- and γ-PCCH, which can be formed by LinA from α-and γ-HCH, respectively, were also converted by LinB. Incubation of such substrates with Escherichia coli BL21 expressing functional LinB originating from Sphingobium indicum B90A showed that both β-PCCH and γ-PCCH were direct substrates of LinB. Furthermore, we identified the main metabolites as 3,4,5,6-tetrachloro-2-cyclohexene-1-ols and 2,5,6-trichloro-2-cyclohexene-1,4-diols by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. In contrast to α-HCH, γ-HCH was not a substrate for LinB. On the basis of our data, we propose a modified γ-HCH degradation pathway in which γ-PCCH is converted to 2,5-cyclohexadiene-1,4-diol via 3,4,5,6-tetrachloro-2-cyclohexene- 1-ol and 2,5,6-trichloro-2-cyclohexene-1,4-diol.

Full text of DOI:10.1021/jf800465q

6594 J. Agric. Food Chem. 2008, 56, 6594–6603
New Metabolites in the Degradation of r- and
γ-Hexachlorocyclohexane (HCH):
Pentachlorocyclohexenes Are Hydroxylated to
Cyclohexenols and Cyclohexenediols by the
Haloalkane Dehalogenase LinB from Sphingobium
indicum B90A
VISHAKHA RAINA,†,‡ DANIEL RENTSCH,§ THOMAS GEIGER,§ POONAM SHARMA,|
HANS RUDOLF BUSER,⊥ CHRISTOF HOLLIGER,# RUP LAL,| AND
HANS-PETER E. KOHLER*,‡
Swiss Federal Institute of Aquatic Science and Technology, Eawag, CH-8600 Du¨bendorf, Switzerland,
Laboratory for Functional Polymers, Empa, Swiss Federal Laboratories for Materials Testing and
Research, CH-8600 Du¨bendorf, Switzerland, Department of Zoology, University of Delhi,
Delhi-110007, India, Swiss Federal Research Station, Agroscope ChanginssWa¨denswil,
CH-8820 Wa¨denswil, Switzerland, and EPFL, ENAC-ISTE, Laboratory of Environmental
Biotechnology, CH-1015, Lausanne, Switzerland
Technical hexachlorocyclohexane (HCH) and lindane are obsolete pesticides whose former production
and use led to widespread contaminations posing serious and lasting health and environmental risks.
Out of nine possible stereoisomers, R-, ꢀ-, γ-, and δ-HCH are usually present at contaminated sites,
and research for a better understanding of their biodegradation has become essential for the
development of appropriate remediation technologies. Because haloalkane dehalogenase LinB was
recently found responsible for the hydroxylation of ꢀ-HCH, δ-HCH, and δ-pentachlorocyclohexene
(δ-PCCH), we decided to examine whether ꢀ- and γ-PCCH, which can be formed by LinA from R-
and γ-HCH, respectively, were also converted by LinB. Incubation of such substrates with Escherichia
coli BL21 expressing functional LinB originating from Sphingobium indicum B90A showed that both
ꢀ-PCCH and γ-PCCH were direct substrates of LinB. Furthermore, we identified the main metabolites
as 3,4,5,6-tetrachloro-2-cyclohexene-1-ols and 2,5,6-trichloro-2-cyclohexene-1,4-diols by nuclear
magnetic resonance spectroscopy and gas chromatography-mass spectrometry. In contrast to
R-HCH, γ-HCH was not a substrate for LinB. On the basis of our data, we propose a modified γ-HCH
degradation pathway in which γ-PCCH is converted to 2,5-cyclohexadiene-1,4-diol via 3,4,5,6-
tetrachloro-2-cyclohexene-1-ol and 2,5,6-trichloro-2-cyclohexene-1,4-diol.
KEYWORDS: HCH; LinA; LinB; dehydrochlorinase; halidohydrolase; Sphingobium indicum B90A; ha-
loalkane dehalogenase; dehydrohalogenation; hydroxylation; pentachlorocyclohexene; PCCH; tetrachlo-
rcyclohexenol; trichlorocyclohexenediol
INTRODUCTION
benzene under suitable conditions leads to a mixture of isomers
and congeners. Theoretically, there are nine HCH stereoisomers,
including one pair of enantiomers (R-HCH), but only one
isomer, γ-HCH, possesses insecticidal properties. The technical
mixture, consisting mainly of R-, ꢀ-, γ-, δ-, and ε-HCH (1),
was introduced as a pesticide in the 1940s and then widely used
in agriculture and for malaria control (2). Later, it was partially
replaced by pure γ-HCH (lindane) and eventually was banned
in most countries because of the environmental persistence of
some of the isomers. However, lindane is enriched from
technical mixtures by fractional crystallization, resulting in large
amounts of isomeric waste, some of which has been dumped
Hexachlorocyclohexane (HCH) was one of the most popular
organochlorine pesticides. Its production by chlorination of
* To whom correspondence should be addressed. Telephone: +41-
44-823-55-21. Fax: +41-44-823-55-47. E-mail: kohler@eawag.ch.
† Present address: KIIT School of Biotechnology, Campus 11,
Bhubaneswar-751024, Orissa, India.
‡ Swiss Federal Institute of Aquatic Science and Technology.
§ Swiss Federal Laboratories for Materials Testing and Research.
| University of Delhi.
⊥ Swiss Federal Research Station.
# EPFL.
10.1021/jf800465q CCC: $40.75  2008 American Chemical Society
Published on Web 07/04/2008

New Metabolites in the Degradation of R- and γ-HCH
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6595
Scheme 1. (Top) Degradation Pathway of δ-HCH and δ-PCCH with the Formation of Hydroxylated Metabolites by LinB (Absolute Stereochemistry of
δ-PCCH and the Following Products D3 and D4 Is Shown Arbitrarily) and (Bottom) Structures and Numbering of Carbon Atoms Used for NMR Signal
Assignments of PCCHs and Hydroxylated Metabolites
over the past 50 years (3). In fact, γ-HCH is currently under
review for addition to the Stockholm Convention on persistent
organic pollutants (POPs) (http://epa.gov/oppt/ar/20052006/
managing/stockholm.htm). Nevertheless, lindane continues to
be produced and still has restrictive use in some developing
countries.
LinB (5, 12, 13). Because δ-PCCH was a good substrate for
LinB, we decided to examine whether ꢀ- and γ-PCCH, which
can be formed by LinA from R- and γ-HCH, respectively, were
also converted by LinB. Here, we document that, both ꢀ- and
γ-PCCH, are direct substrates of LinB from strain B90A,
identify the reaction products, and propose a modification of
the established γ-HCH degradation pathway.
Several strains of the genus Sphingobium (previously Sph-
ingomonas) with the ability to degrade HCH under aerobic
conditions were isolated and identified. These strains originated
from different geographical locations, such as Japan (Sphingo-
bium japonicum UT26), India (Sphingobium indicum B90A),
and France (Sphingobium francense Sp+) (4), and slight
variations in the degradation of various HCH isomers were
observed. Among these strains, S. japonicum UT26 is studied
best in terms of the metabolic pathway of γ-HCH and the
putative lin genes involved (5). It is widely accepted that the
three enzymes LinA, LinB, and LinC catalyze the reactions of
the upper pathway and that LinD, LinE, and LinF catalyze those
of the lower one. It is suggested that LinA converts γ-HCH to
1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) in two de-
hydrochlorination steps with γ-pentachlorocyclohexene (γ-
PCCH) as an intermediate and that LinB acts on 1,4-TCDN to
form 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL) and
subsequently 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-
DDOL) (5). However, it should be emphasized that up to date
neither 1,4-TCDN nor 2,4,5-DNOL were actually isolated and
rigorously identified, because of their alleged inherent instability.
Recently, different LinB enzymes were found to be respon-
sible for the conversion of ꢀ- and δ-HCH into pentachlorocy-
clohexanols and tetrachlorocyclohexanediols (6–9). Furthermore,
we and others were able to show that δ-HCH was metabolized
to 3,4,5,6-tetrachloro-2-cyclohexene-1-ol (D3) and 3,5,6-trichloro-
2-cyclohexene-1,4-diol (D4) via δ-PCCH in the presence of
LinA and LinB from strains B90A and BHC-A and that
δ-PCCH was a good substrate for LinB (Scheme 1) (10, 11).
LinB is an enzyme with a broad substrate range. However,
metabolites formed during the degradation of HCH isomers
other than δ-PCCH have not yet been tested as substrates for
MATERIALS AND METHODS
Chemicals and Reference Compounds. Pure R-, γ- and δ-HCH
(98-99%, respectively) were obtained from Riedel-de-Hae¨n (Seelze,
Germany). γ- and δ-PCCH were chemically synthesized from γ- and
δ-HCH by alkaline dehydrochlorination according to published pro-
cedures (14), ꢀ-PCCH was synthesized from R-HCH according to ref
1. Typically, reaction products were percolated through silica gel (silica
gel 60, 230-400 mesh; Merck, Darmstadt, Germany; glass column;
25 cm × 2 cm i.d.) with gradient elution of hexane/diethyl ether
mixtures of 100:0 (100 mL), 40:1 (100 mL), and 20:1 (100 mL). The
combined extracts were washed with equal volumes of dilute HCl, 5%
NaHCO3 solution, and water and finally dried over a column of
anhydrous Na2SO4. The purities of the PCCHs were established by
gas chromatography-mass spectrometry (GC-MS), and the relative
stereochemistries were established by 1D and 2D nuclear magnetic
resonance (NMR) spectrometry (see below). Whereas both, γ- and
δ-PCCH were pure (>99%), the synthesized ꢀ-PCCH contained
additional PCCHs and remaining starting material (ꢀ-PCCH mixture
1; structures and typical sample composition, see the Results). For some
resting cell assays (see below), this crude reaction product was further
purified by flash chromatography (see the Supporting Information),
resulting in a mixture of ꢀ-PCCH and θ-PCCH (∼80:20) (ꢀ-PCCH
mixture 2). PCCHs are chiral, and all of the chemically synthesized
material was used as racemate.
Cloning of linB into Expression Vector pET-3c. To clone linB in
an expression vector, we constructed polymerase chain reaction (PCR)
primers using already known gene sequences of B90A from the database
of the National Center for Biotechnology Information (NCBI, Bethesda,
MD) as described in ref 10.
Resting Cell Assay with Various Clones and Extraction of
Metabolites. A total of 500 mL of LB medium was inoculated with
an overnight seed culture (1% v/v) of Escherichia coli BL21-pET-3c

6596 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Raina et al.
1
Table 1. 1H and 13C Chemical Shifts and H,1H Coupling Constants of ꢀ-, γ-, δ-, θ-, and η-PCCH in Benzene-d6
δ (1H) (ppm) and J (1H,1H) (Hz)
δ (13C) (ppm)
δ-PCCH
positiona
ꢀ-PCCHb
γ-PCCHc
δ-PCCH
θ-PCCHb,d
η-PCCHc,d
ꢀ-PCCHb
γ-PCCHc
θ-PCCHb,d
η-PCCHc,d
1
2
3
4
5
6
133.6
127.0
55.8
59.4
62.8
63.3
130.7
128.0
57.7
59.7
62.3
59.8
131.4
128.1
58.5
63.8
64.8
63.0
132.3
127.2
60.1
62.0
60.1
62.1
132.6
127.9
58.6
57.8
58.5
63.6
5.28
3.68
3.02
4.38
6.34
5.07
4.83
5.04
5.18
J23 ) 3.5
5.33
3.59
3.32
3.56
5.19
3.80
4.20
2.98
3.83
J23 ) 3.1
6.43
5.21
4.86
4.95
5.19
J23 ) 5.7
4.04
3.87
J23 ) 6.3
J26 ) 1.3
J34 ) 3.7
J36 ) 0.8
J45 ) 10.8
J56 ) 7.4
J23 ) 2.3
J26 ) 1.6
J34 ) 8.4
J36 ) 2.8
J45 ) 11.1
J56 ) 8.0
J34 ) 7.6
J34 ) 8.2
J34 ) 3.9
J45 ) 2.6
J56 ) 4.0
J45 ) 11.5
J56 ) 3.6
J45 ) 11.5
J56 ) 3.9
a For atom numbers, see Scheme 1. b The three pairs of 1H/13C signals are assigned to the starting material R-HCH: 3.57/62.3, 4.21/59.0, and 3.90/64.1. c In DMSO-d6
solution. d θ- and η-PCCH correspond to the compounds mentioned as X1 and X2 in ref 16.
Scheme 2. Structures of PCCHs Formed from R-HCH by Different 1,2
HCl Elimination Reactions (Absolute Stereochemistry Shown Arbitrarily;
the Reactions Were Carried out with Racemic R-HCH): (a) trans-Diaxial
Elimination of H-3a/Cl-2a, (b) cis Elimination of H-2e/Cl-1a, (c) trans
Elimination of H-2e/Cl-3e, Corresponding to trans-Diaxial Elimination of
H-2a/Cl-3a for the Inverted R-HCH Conformer II in Equilibrium, (d) cis
Elimination of H-4a/Cl-3e or H-3a/Cl-4e (/, Second Reaction Results in
the Other Enantiomer of γ-PCCH), and (e) cis Elimination of H-4a/Cl-5e
(containing linB). The culture was incubated at 37 °C and 200 rpm
until it reached an OD600 ∼ 0.6–0.8. At this point, the culture was
induced with isopropyl-ꢀ-D-thiogalactopyranosid (IPTG) at a final
concentration of 0.5 mM and further incubated at 37 °C and 200 rpm.
After 4 h, the culture was harvested and washed twice with potassium
phosphate buffer (10 mM; pH 7) and the culture pellet (∼0.3 mg/mL
dry weight) was resuspended in the same amount (500 mL) of buffer.
To this suspension, ꢀ-, γ-, or δ-PCCH was added separately and
incubated at 30 °C at 200 rpm (appropriate incubation times were
chosen between 1 and 20 h). Subsequently, the resting cells were
extracted twice with equal volumes of ethyl acetate. The combined
extracts were dried over Na2SO4 (Fluka, Buchs, Switzerland) and
percolated through silica gel (230-400 mesh), and the solvent
evaporated at 40 °C. The dry extracts were subjected to NMR analysis,
and small sample aliquots were dissolved in hexane or ethyl acetate
for GC-MS. Control experiments were performed as described in ref
10.
GC-MS Analysis. The samples were analyzed on a VG Tribrid
double-focusing magnetic sector hybrid mass spectrometer (VG Analytical,
Manchester, U.K.) operated under electron ionization conditions (EI; 50
eV; ion source, 180 °C), using a 30 m BGB-5 capillary column (0.32 mm
i.d.; film thickness, 0.25 µm; BGB Analytik, Adliswil, Switzerland). This
column showed very similar column characteristics as the earlier used SE54
or DB-5 MS columns (10). It allowed the samples to be analyzed without
prior derivatization (acetylation), resulting in peaks with acceptable tailing
of the polar metabolites. Samples were injected in ethyl acetate at 70 °C,
and the column was temperature-controlled as follows: 70 °C, 2 min
isothermal, 20 °C/min to 120 °C, and then 5 °C/min to 280 °C, followed
by an isothermal hold at this temperature. Retention time measurements
were started at 120 °C and reported relative to those of the n-alkanes as
retention indices (RIs; e.g., RI ) 1800 for n-octadecane), using linear
interpolation in the temperature-programmed runs. Some samples were
re-analysed after acetylation, which was carried out as reported earlier
(10).
NMR Analysis. Stereochemical information was obtained from 1H
and 13C NMR spectra recorded at 400.13 (100.61) MHz on a Bruker
Avance-400 NMR spectrometer (Bruker Biospin AG, Fa¨llanden,
Switzerland). The 1H and 13C NMR spectra and the 1H,13C 2D
correlation experiments were performed at 298 K using a 5 mm
broadband inverse probe with z gradient (100% gradient strength of
53.5 G cm-1) and 90° pulse lengths of 6.8 µs (1H) and 14.9 µs (13C).
All spectra were recorded with the Bruker standard pulse programs
and parameter sets, and the 1H/13C chemical shifts were referenced
internally using the resonance signals of acetone-d6 at 2.05/29.8 ppm,
CDCl3 at 7.26/77.0 ppm, benzene-d6 at 7.15/128.0 ppm, and DMSO-
d6 at 2.49/39.5 ppm.
limited NMR spectroscopic data exist for PCCHs (14, 15), we
undertook a systematic study of the various PCCHs to determine
the exact stereochemistry of the compounds. In Table 1, the
NMR data (chemical shifts and coupling constants) of the five
PCCHs used as substrates for degradation experiments with
LinB are summarized, and their structures are shown in Scheme
2. Note that the reactions were carried out with racemic R-HCH,
but to simplify matters, only reactions with (+)-R-HCH are
shown in the schemes. The numbering of carbon atoms used
for the NMR signal assignments of PCCHs and metabolites are
depicted in Scheme 1. Besides reporting NMR data for ꢀ-, γ-,
and δ-PCCH, we also report data for two additional PCCHs
that we observed as minor products when R-HCH was chemi-
cally dehydrohalogenated. The two PCCHs were named θ- and
η-PCCH because they can be envisioned to form by trans-diaxial
RESULTS
Stereochemical and Conformational Analysis of Pen-
tachlorocyclohexenes (PCCHs) Produced from Different
HCH Isomers by Chemical Dehydrogenation. Because only

New Metabolites in the Degradation of R- and γ-HCH
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6597
Scheme 3. Degradation Pathway of R-HCH and ꢀ-, θ-, and η-PCCH (Reaction Scheme Arbitrarily Shown for (+)-R-HCH and Its PCCHs)a
a Compounds in parentheses were not isolated or characterized.
dehydrohalogenation of θ- and η-HCH, respectively. They had
been observed earlier but were not further characterized (1, 16).
In the following, we abbreviate the metabolites from ꢀ- and
γ-PCCH as A (from R-HCH, the precursor of ꢀ-PCCH) and G
compounds, respectively, and we abbreviate those from θ- and
η-PCCH as T and I compounds, respectively. We already have
introduced the B and D compounds as metabolites of ꢀ- and
δ-HCH, respectively (10).
some ꢀ-, δ-, θ-, and η-PCCH (typical composition 78:17:3:
2) and traces of γ-PCCH besides remaining R-HCH (ca. 40%)
(the relative amounts corresponded to those determined earlier
by GC-MS (1)). For γ-PCCH, a J34 value of 7.6 Hz showed
that the pair of protons H-3 and H-4 had the relative
configuration pseudo-axial-pseudo-axial. Therefore, H-5
must be located in pseudo-equatorial position (J45 ) 2.6 Hz).
ꢀ-PCCH was the major dehydrochlorination product of
R-HCH. The formation of additional PCCHs requires some
consideration. In Scheme 2, we show the structures of the
five PCCH that can theoretically be formed by a 1,2
elimination of HCl from R-HCH. Owing to the C2 symmetry
axis through the carbon bonds C-1/C-2 and C-4/C-5, the
positions 1 and 2, 3 and 6, and 4 and 5 are equivalent. From
vicinal pairs of H and Cl atoms, a total of six HCl
eliminations can be envisioned. The major product ꢀ-PCCH
confirms that a trans-diaxial HCl elimination from the more
stable conformer I (two axial Cl atoms) is favored. With a
cis H(eq)/Cl(ax) elimination, the formation of δ-PCCH could
be explained, and a trans H(eq)/Cl(eq) elimination or more
likely an alternate trans-diaxial HCl elimination of the
R-HCH conformer II (four Cl atoms in axial position) would
be a plausible explanation for the formation of θ-PCCH. The
formation of γ- and η-PCCH possibly result from a cis H(ax)/
Cl(eq) elimination. However, γ-PCCH was only observed
in very small amounts (<0.3%) and could also be formed
from conformer II by a cis H(eq)/Cl(ax) elimination.
Degradation of ꢀ-PCCH by Clones of E. coli Expressing
LinB from S. indicum B90A. Resting cell incubations with E.
coli expressing LinB were carried out with ꢀ-PCCH mixtures
1 and 2. The major product observed by GC-MS was a
trichlorocyclohexenediol A4 (Scheme 3), apparently a stereo-
isomer of the earlier identified δ-PCCH-metabolite D4 (10).
Although low in concentration, a small peak of the tetrachlo-
rocyclohexenol A3 (an analogue of the δ-PCCH-metabolite D3)
was also detected. In Figure 1, we present EI mass spectra of
A3 and A4 (also included are spectra of the related metabolites
A1 and A2, see the Discussion). As expected, the mass spectra
of A3 and A4 are similar to those of D3 and D4 (10),
respectively. In Table 2a, we report the RIs for the A
metabolites. When samples were acetylated and re-analyzed,
the corresponding mono- and diacetates were observed with
Dependent upon the solvent, the overlap of resonances was
observed (1H and/or 13C NMR spectra), inhibiting appropriate
signal assignment and/or exact determination of 1H,1H
coupling constants. Thus, the determination of the relative
conformation of the hydrogen atoms in the molecules often
failed, and, e.g., for η-PCCH, the coupling constant J56 was
determined indirectly from a 1D TOCSY experiment. Owing
to coalescence effects, in the case of γ-PCCH, some 1H NMR
signals were significantly broadened and incomplete resolu-
tion of the coupling pattern in the 1H NMR spectra was
observed. The trans-diaxial configurations of H-4 and H-5
of ꢀ-, δ-, η-, and θ-PCCH were established with J45 ≈ 11
Hz (Table 1), whereas for the γ isomer, the dihedral angle
between these protons must be almost 90° (J45 ) 2.6 Hz).
From the magnitudes of J34 and J56, the relative conformations
of H-3 and H-6 with respect to H-4 and H-5 were derived.
Supplementary NMR experiments supported the assignment
of the relative stereochemistry. While for ꢀ-PCCH, a nuclear
Overhauser enhancement (NOE) between H-4 and H-6
indicated that these protons must be located at the same side
of the molecular plane; the absence of a NOE between H-6
and H-3 pointed to a pseudo-equatorial configuration of H-3
(this was further supported by J34 ) 3.7 Hz). Also, for
δ-PCCH, a NOE was found between H-4 and H-6, and from
the magnitudes of the coupling constants (J34 ) 8.4 Hz, J45
) 11.1 Hz, and J56 ) 8.0 Hz), we concluded that the
neighboring protons in the saturated part of the ring must be
located trans to each other (Schemes 1 and 2). The significant
deviation in the magnitudes of J56 between δ-PCCH (8.0 Hz)
and θ-PCCH (3.6 Hz) resulted from the opposite configu-
ration of the chlorine atom at C-6 (Table 1 and Scheme 2).
Similarly, ꢀ-PCCH and its epimer η-PCCH differed in the
magnitude of J56 (7.4 and 3.9 Hz). It must be noted that
chemically synthesized batches of ꢀ-PCCH always contained

6598 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Raina et al.
Figure 1. EI mass spectra of metabolites A1-A4.
Table 2. Retention and EI MS Data of (a) R-HCH/ꢀ-PCCH and (b) γ-HCH/γ-PCCH Metabolites
compounda
type of compound
RI
EI MS datab
(a) Retention and EI MS Data of R-HCH/ꢀ-PCCH Metabolites
R-HCH
ꢀ-PCCH
θ-PCCH
η-PCCH
δ-PCCH
γ-PCCH
A1
A2
A3
A4
T4 and I4
hexachlorocyclohexane
pentachlorocyclohexene
pentachlorocyclohexene
pentachlorocyclohexene
pentachlorocyclohexene
pentachlorocyclohexene
pentachlorocyclohexanol
tetrachlorocyclohexanediol
tetrachlorocyclohexenol
trichlorocyclohexenediol
trichlorocyclohexenediols
1696
1591
1565
1607
1533
1460
1753
1804
1535
1564
1553
288
252
252
252
252
252
270, Cl5; 235, 156 (RDA), 125, 109; see Figure 1a
(252); 217, 199, 181, 138 (RDA, weak), 91; see Figure 1b
(234); 199, 163, 138 (RDA); see Figure 1c
(216); 180/181, 163, 145, 120 (RDA), 91; see Figure 1d
(216); 180/181; 163; 145; 120 (RDA); 91
(b) Retention and EI MS Data of γ-HCH/γ-PCCH Metabolites
γ-HCH
γ-PCCH
2,5-DDOL
2,6-DDOL
G3
hexachlorocyclohexane
pentachlorocyclohexene
dichlorocyclohexadienediol
dichlorocyclohexadienediol
tetrachlorocyclohexenol
trichlorocyclohexenediol
tetrachlorocyclohexenol
1758
1460
1429
1454
1532
1628
1558
288
252
180, 178, 162, 145; see Figure 2d
180, 178, 162, 145
(234); 199, 163, 138 (RDA); see Figure 2a
(216); 180/181, 163, 145, 120 (RDA), see Figure 2c
234, Cl4; 199, 163, 138 (RDA); see Figure 2b
G4
G3b
a For structures, see Schemes 1–4. b EI MS data are reported as follows: all ions, monoisotopic, first number, molecular ion (not observed if in parentheses), followed
by important fragment ions.
appropriate shifts in retention times and masses (data not
shown). Further, traces of a dichlorophenol (M+ ) 162, Cl2)
and a trichlorobenzene (M+ ) 180, Cl3) were also detected.
The relative stereochemistry of metabolite A3 was established
experiments (Table 3). The main deviation between A3 and
A4 (note the different numbering of the carbon atoms in Scheme
1) concerned J45 ) 8.0 Hz (A3) and J16 ) 3.4 Hz (A4), whereas
all other coupling constants were of the same magnitude. This
again indicates that the OH group at C-1 of A4 was introduced
in a SN2 type of reaction (Scheme 3). The relative configuration
of an additional trichlorocyclohexenediol (T4) was established
by NMR experiments (Table 3 and Scheme 3), with material
extracted from incubations with ꢀ-PCCH mixture 2.
E. coli expressing LinB was able to transform all five PCCHs
present in ꢀ-PCCH mixture 1. From GC-MS data, we conclude
that several bis-hydroxylated metabolites were present. As
expected, one additional metabolite was identified as the already
known compound D4 [identical 1H and 13C chemical shifts and
1
from the magnitudes of the relevant H,1H coupling constants
(8.2, 11.5, and 8.0 Hz; Table 3). Thus, each of the protons H-1,
H-6, H-5, and H-4 must be located in pseudo-axial position with
H-4 and H-6 at one and H-1 and H-5 at the opposite side of the
ring plane. Taking into account the stereochemistry of the
substrate ꢀ-PCCH, we conclude that the OH group at C-1 of
A3 was introduced in a SN2 type of reaction, as observed earlier
for the corresponding metabolites from δ-PCCH.
The relative stereochemistry of the main component A4 was
established from the scalar coupling constants observed in NMR

New Metabolites in the Degradation of R- and γ-HCH
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6599
Scheme 4. Degradation Pathway of γ-HCH and γ-PCCH (Absolute Stereochemistry Shown Arbitrarily)
1
Table 3. 1H and 13C Chemical Shifts and H,1H Coupling Constants of Tetrachlorocyclohexeneols and Trichlorocyclohexenenediols in Benzene-d6
δ (1H) (ppm) and J (1H,1H) (Hz)
δ (13C) (ppm)
positiona
A3
A4
G3
G3bb
G4b
T4
I4
A3
A4
G3
G3bb
G4b
T4
I4
1
2
3
4
5
6
3.38
5.46
3.56
3.48
5.41
4.45
4.30
3.68
3.60
71.1
73.2
66.7
130.2 132.1 129.6
69.5
nd
70.4 74.9
134.2 136.1
74.7
nd
5.49
3.56
3.90
3.11
1.97
1.58
6.06
4.82
4.41
4.77
5.92
4.59
4.61
4.69
4.49
5.44
3.56
3.26
4.08
2.00
1.65
5.45
3.50
3.35
3.53
130.3 129.0 130.3 124.4 128.6 125.6 127.7
4.04
3.70
3.16
4.34
3.68
3.85
1.85
64.0
64.5
65.2
71.6
62.5
62.4
61.7
62.2
62.6
57.3
61.3
62.2
68.8
63.5
60.8
66.4
66.4
62.0
72.0
65.5
64.6
c
OH-1
OH-4
c
J24 ) 1.4
J14 ) 2.7
J12 ) 2.2
J16 ) 8.2
J56 ) 11.5 J56 ) 11.5
J45 ) 8.0 J16 ) 3.4
J1-OH ) 10.2 J13 ) 1.4 J1-OH ) 8.6
J14 ) 1.8
J12 ) 2.9
J16 )3.7
J56 ) 2.3
J45 ) 6.5
J14 ) 2.0
J34 ) 3.7
J45 ) 6.3
J56 ) 2.4
J16 ) 4.4
J13 ) 0.9
J34 ) 3.1
J45 ) nd
J56 ) 2.0
J16 ) 4.9
J13 ) 1.4
J34 ) 5.3
J45 ) 3.3
J56 ) 10.1 J56 ) 11.6
J16 ) 6.6 J16 ) 8.1
J13 ) 1.7
J34 ) 1.7
J45 ) 8.1
J34 ) 2.6
J45 ) 7.7
a For atom numbers, see Scheme 1. b G3b measured in CDCl3 and G4 measured in acetone-d6 solution. c All other OH resonances were broad (and not assignable)
or not detected.
1
scalar coupling constants (10)]. In the H NMR spectra, the
Degradation of γ-PCCH by Clones of E. coli Expressing
LinB from S. indicum B90A. Incubation of γ-PCCH with E.
coli expressing LinB lead to the formation of several metabo-
lites; the EI mass spectra of the most prominent ones are
presented in parts a-d of Figure 2. The major metabolite
observed by GC-MS was the tetrachlorocyclohexenol G3,
which is a stereoisomer of D3 and A3 but eluted somewhat
earlier (see Table 2b for RI values). Smaller amounts of further
tetrachlorocyclohexenols and a trichlorocyclohexenediol (G4)
were also detected. The prominent peak of a dichlorophenol
signals of at least one additional compound could be detected.
Although the correlations observed in the H,13C 2D NMR
1
experiments were quite weak, the chemical shifts and the
stereochemistry of I4 could be deduced. The relevant coupling
constants found for I4 and δ-PCCH (Tables 1 and 3) indicate
that the two compounds have the same relative stereochemistry.
Typically, an overnight incubation of ꢀ-PCCH mixture 1
resulted in complete elimination of all PCCHs. Besides remain-
ing R-HCH, these reaction mixtures contained A4, D4, T4, and
I4 (typical composition 58:10:3:2). Furthermore, detailed analy-
sis of the aromatic region of the 1H NMR spectra revealed the
presence of small amounts of 1,2,4-trichlorobenzene and 2,5-
dichlorophenol. These data also clearly show that PCCHs were
more rapidly converted than R-HCH. Incubation of pure R-HCH
with E. coli expressing LinB mainly led to the formation of A4
and T4 (see the Discussion).
(M+ ) 162, Cl2) and small peaks of trichlorophenols (M+
)
196, Cl3), chlorophenol (M+ ) 128, Cl), 2,5-dichlorohydro-
quinone (M+ ) 178, Cl2), and trichlorobenzenes (M+ ) 180,
Cl3) were also observed. Upon acetylation of appropriate
samples, the corresponding mono- and diacetates were observed,
with the appropriate shifts in retention time and masses (data
not shown).

6600 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Raina et al.
Figure 2. EI mass spectra of γ-PCCH metabolites.
1
Table 4. 1H and 13C Chemical Shifts and H,1H Coupling Constants of
Dichlorocyclohexadienediols (DDOL) in Benzene-d6
The identification of 2,5- and 2,6-dichloro-2,5-cyclohexadi-
ene-1,4-diol (2,5- and 2,6-DDOL) by GC-MS was not straight-
forward (see spectrum for 2,5-DDOL in Figure 2d) because
molecular ions (M+) at m/z 180 are not easily recognized.
However, the fragment ions (monoisotopic) at m/z 178, 162,
and 145, interpreted as M+-2H, M+-H2O, and M+-Cl,
respectively, and the formation of a diacetate eventually
confirmed this identification (data not reported). In the 1H NMR
spectra of samples from γ-PCCH incubation experiments with
E. coli expressing LinB, the resonances of five unsaturated
metabolites were observed. These metabolites were identified
as G3, G3b, G4, 2,5-DDOL, and 2,6-DDOL (Scheme 4).
δ (1H) (ppm) and J (1H,1H) (Hz)
δ (13C) (ppm)
positiona
2,5-DDOLb
2,6-DDOL
3.85
2,5-DDOL
2,6-DDOL
1
2
3
4
3.85
66.7
135.2
126.2
68.4
133.6
127.0
64.2
5.63
5.67
3.9
J16 ) 3.8
J34 ) 4.0
a For atom numbers, see Scheme 1. b δ (1H) of 4.56 and 6.14 ppm observed
in CDCl3 solution corresponds to earlier findings (17).
1
In the case of G3, it is evident from the H,1H coupling
constants that H-4 and H-5 are in axial and pseudo-axial
positions (J45 ) 6.5 Hz, Table 3), whereas from J56 ) 2.3 Hz
and J16 ) 3.7 Hz, we conclude that the relative stereochemistry
indicated in Scheme 4 must be correct (for atom numbering,
see Scheme 1). In benzene solution, NOEs were observed from
H-5 f H-1 and from H-1 f H-2, H-5, and H-6. This proved
that H-1 and H-5 are on the same side of the cyclohexane ring
plane and H-4 is on the opposite side. Again, the data indicate
that the Cl/OH substitution reaction from γ-PCCH to G3
proceeded in a SN2-type reaction under inversion of the
configuration at C-3.
When the metabolite mixture was fractionated by flash
chromatography, we characterized an additional metabolite
(G3b) in the fraction containing mainly G3 (G3/G3b ≈ 97/3).
The relative stereochemistry at carbons 1, 4, 5, and 6 in G3b
was readily established from the coupling constants observed,
and the DQF-COSY and 1H,13C correlation experiments
revealed that the carbon bearing the OH group must be next to
the “quaternary” (nonhydrogenated) carbon at the double bond.
Therefore, the minor metabolite G3b was most likely formed
by hydroxylation of γ-PCCH at C-6. This is in contrast to the
general route observed for the other cyclohexenols, which
always started with hydroxylation at C-3.
1
Two further products showing H signals at 5.63 and 5.67
1
ppm revealed 3 and 4 carbon resonances only in the H,13C
correlated spectra (Table 4). Therefore, these signals must have
originated from symmetric molecules (assuming six-membered
1
carbon rings). The H NMR data of one of the products, 2,5-
DDOL, correspond to earlier findings (17).
Only traces of the intermediate product G4 were detected by
NMR spectroscopy. Nevertheless, evidence for the relative
configuration of G4 was obtained from the 2D NMR experi-
ments of a purified fraction containing mainly DDOLs and G4
(flash chromatography; see the Supporting Information). From
the NMR data, it is evident that H-5 and H-6 have a dihedral
angle close to 90° (J56 ) 2.0 Hz), and J16 ) 4.9 Hz also pointed
to a cis configuration of H-1 with respect to H-6. Unfortunately,
J45 was not resolved, and only weak correlations over 2-3 bonds
wereobservedalsowithlongermixingtimesintheHSQC-TOCSY
spectrum. Assuming the relative stereochemistry for G4 as

New Metabolites in the Degradation of R- and γ-HCH
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6601
Scheme 5. Proposed Reaction Mechanism for the Formation of Metabolites A4 and T4 from R-HCH by LinB
shown in Scheme 4 (no H atom is trans to the next one), the
absence of correlation signals in the HSQC-TOCSY is
The formation of D4, T4, and I4 in our experiments with
ꢀ-PCCH mixture 1 can be explained by sequential SN2-type
Cl/OH exchange reactions of LinB with the additional PCCHs
(δ-, θ-, and η-PCCH) present (Schemes 1 and 3). The small
amounts of 1,2,4-TCB observed are possibly formed by an
alternative pathway with 1,4,5,6-tetrachlorocyclohexa-1,3-diene
as an intermediate product after elimination of Cl-3/H-4 of
ꢀ-PCCH in trans-diaxial positions. The release of either Cl-5
or Cl-6 followed by re-aromatization yields 1,2,4-TCB as a
single product. Finally, the traces of 2,5-dichlorophenol detected
may be formed from the low amount of γ-PCCH present in
this mixture as discussed below.
1
explained by the small H,1H coupling constants. The signals
of H-4 and H-5 were both rather broad without any resolved
coupling, indicating that G4 exists in more than one stable
conformation.
From a typical incubation experiment of γ-PCCH with Lin
B, the isolated product contained 62% G3, 0.9% G3b, 1.4%
G4, 17% 2,5-DDOL, 3.7% 2,6-DDOL, 13% 2,5-DCP, 0.7% 3,5-
DCP, 0.7% 3,4-DCP, and trace amounts of other aromatic
compounds. After extended storage of the metabolite mixture
in the absence of biological active media, 1.6% of 2,6-DCP
was also observed. Concomitant to the formation of 2,6-DCP,
the amounts of 2,6-DDOL decreased to 0.5%.
NMR analysis of incubations of R-HCH with LinB from
S. indicum B90A revealed the presence of A4 and T4. Other
metabolites were not detected by NMR. These observations
are surprising, because we did not observe HCl elimination
products (cyclohexenols) in incubations of ꢀ- and δ-HCH
with E. coli expressing LinB (10), and it was clearly
established that D3 and D4 were the products of hydroxy-
lations of δ-PCCH and not of HCl eliminations of D1 and
D2, respectively. As shown in Scheme 5, we propose that
A4 and T4 are formed by HCl elimination of hydroxylated
metabolites of R-HCH. In contrast to the metabolites D1,
D2, B1, and B2 described earlier (10), the putative metabo-
lites A1a, A1c, and A2 have trans-diaxial H/Cl arrangements
in R,ꢀ position relative to the hydroxy group, and therefore,
trans-diaxial HCl elimination is a reasonable and perhaps
favorable reaction for these compounds. Although not
detected by NMR, trace amounts of A1 and A2 (see Table
2a and parts a and b of Figure 1) were detected by GC-MS
when R-HCH was incubated with E. coli expressing LinB.
This further supports the mechanism proposed in Scheme 5.
However, presently, it is not known whether the proposed
HCl eliminations occur spontaneously or whether they are
enzyme-catalyzed.
Surprisingly and in contrast to the observations for R-, ꢀ-,
and δ-HCH, no metabolites were formed when pure γ-HCH
was incubated with E. coli expressing LinB.
DISCUSSION
In our previous studies, we showed that LinB from S. indicum
B90A converted ꢀ- and δ-HCH into pentachlorocyclohexanols
(B1 and D1) and tetrachlorocyclohexanediols (B2 and D2) (7).
LinB also converted δ-PCCH to the tetrachlorocyclohexenol
D3 and the trichlorocyclohexenediol D4 (see Scheme 1 for the
products of δ-HCH and δ-PCCH) (10). The pentachlorocyclo-
hexanols B1 and D1 were both formed by SN2-type reactions,
with replacements of equatorial chlorines in the parent HCHs.
In the present study, we show that LinB from S. indicum
B90A readily metabolizes ꢀ- and γ-PCCH. The tetrachlorocy-
clohexenols (A3 and G3) as well as the trichlorocyclohexene-
diols (A4 and G4) were isolated and characterized; both pairs
are constitutional isomers of D3 and D4, respectively. Each
metabolite shows a well-defined stereochemistry that agrees with
formation by SN2-type mechanisms, as was observed with
δ-PCCH (10). Apparently, the Cl/OH replacement takes place
mainly at C-3 of a PCCH, irrespective of whether the chlorine
in allylic position is in pseudo-equatorial (δ-PCCH) or pseudo-
axial (ꢀ-PCCH) position. Only in the case of γ-PCCH, some
Cl/OH exchange at the alternative allylic position C-6 was
observed (metabolite G3b) but, notably, still following a SN2
mechanism.
Our data clearly show that γ-PCCH served as a direct
substrate for LinB from S. indicum B90A, forming the
hydroxylated metabolites G3 and G4. Further metabolites,
such as 2,5-DDOL, 2,6-DDOL, and 2,5-, 2,6-, and 3,5-
dichlorophenols were also detected. Scheme 4 shows a
reaction scheme that explains the formation of these me-

6602 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Raina et al.
Scheme 6. Formation of 2,5- and 2,6-DDOL by trans-Diaxial HCl
Eliminations of Two Conformers of γ-HCH Metabolite G4
amino acids, respectively, and the LinB enzymes of strain
Sp+ and strain UT26 differ from each other by three amino
acids (7), this branch might well be operative in other HCH-
degrading strains, because resting cell incubations of wild-
type strains B90A, UT26, and Sp+ also showed degradation
of PCCHs to hydroxylated metabolites (data not shown).
Our data imply that LinB will compete with LinA or LinA1/
A2 for HCHs as well as for PCCHs as substrates in HCH-
degrading bacteria. They further indicate that degradation of
HCH isomers is probably not channeled along a well-defined
pathway but rather ramifies into a network of competing
reactions that possibly lead to a range of chlorinated and
hydroxylated metabolites. More detailed experiments will be
needed to exactly evaluate the formation and further metabolism
of such metabolites in ViVo.
tabolites. It is assumed that both 2,5- and 2,6-DDOL are
formed from G4: elimination of H-5/Cl-6 of G4 will lead to
2,5-DDOL and elimination of H-6/Cl-5 to 2,6-DDOL (see
Scheme 6). Preferably, the HCl elimination reaction is
favored when both atoms are in pseudo-axial positions.
Owing to the neighboring effect of Cl-2, OH-1 probably is
forced into pseudo-equatorial position. Possibly, the confor-
mation of G4 with H-5 and Cl-6 in the pseudo-axial position
is preferred, and hence, 2,5-DDOL is the main product.
Considering the stereochemistry at C-5 and C-6 of G4,
obviously, HCl is readily eliminated, because at the neigh-
boring carbons, a chlorine and a proton are always in trans-
diaxial position. Therefore, elimination reactions are expected
to occur rapidly, explaining why only small amounts of G4
were detected. We were not able to determine the relative
configurations of the hydroxyl groups in 2,5- and 2,6-DDOL.
On the basis of the stereochemistry at G4, cis configuration
of the OH groups is expected (see Scheme 6). Similar
elimination reactions are not expected for A4 or D4, because
trans-diaxial HCl eliminations are not possible (equatorial
chlorine atoms at positions C-5 and C-6). Indeed, incubations
of E. coli expressing LinB with R-HCH, ꢀ-PCCH, or δ-PCCH
gave rise neither to DDOLs nor to dichlorophenols. We can
therefore attribute the stereoselective Cl/OH substitution
reactions to LinB. However, the question whether some of
the subsequent dehydrochlorination or dehydratation reactions
occur non-enzymatically remains unanswered.
At the moment, it is widely agreed that LinA transforms
γ-PCCH to an unstable 1,4-TCDN that then undergoes a two-
step hydrolytic dehalogenation mediated by LinB to form
2,4,5-DNOL and 2,5-DDOL. It is suggested that the me-
tabolites 1,2,4-TCB and 2,5-DCP are formed non-enzymati-
cally from 1,4-TCDN and 2,4,5-DNOL, respectively (5), and
a similar pathway is assumed for the metabolism of R-HCH.
However, it should be emphasized that thus far neither 1,4-
TCDN nor 2,4,5-DNOL was actually detected. Their role in
the metabolism of HCH is solely based on circumstantial
evidence (18). Here, we show that γ-PCCH as well as
ꢀ-PCCH are direct substrates of LinB, and we were able to
isolate and characterize novel metabolites (G3 and G4). We
have to conclude that besides the established reactions, LinB
also catalyzes hydroxylations of γ-PCCH and G3. On the
basis of our data, we suggest an additional branch of the
pathway for the degradation of γ-HCH in S. indicum B90A
(Scheme 4) with hydroxylation reactions catalyzed by LinB
to yield G3 and G4, with the latter subsequently undergoing
dehydrochlorination to 2,5-DDOL and small amounts of 2,6-
DDOL. Although the LinB enzyme of strain B90A differs
from those of strain Sp+ and strain UT26 by six and seven
ACKNOWLEDGMENT
We thank Simon Huber (Empa) for help with flash chromato-
graphy.
Supporting Information Available: 1H,13C HMBC correla-
tions used for NMR shift assignments (Table S1), characteriza-
tion of aromatic compounds, 1H NMR spectra of PCCH isomers
in benzene-d6 (Figure S1), 1H NMR spectra of metabolites from
ꢀ-PCCH incubated with LinB (B90A) measured in C6D6 (Figure
1
S2), H NMR spectra of metabolites from γ-PCCH incubated
with LinB (B90A) in CDCl3 (Figure S3), and samples separated
by flash chromatography, elution conditions, and compositions
of fractions of interest (Table S2). This material is available
free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
(1) Buser, H.-R.; Mu¨ller, M. D. Isomer and enantioselective degrada-
tion of hexachlorocyclohexane isomers in sewage sludge under
anaerobic conditions. EnViron. Sci. Technol. 1995, 29, 664–672.
(2) Li, Y. F. Global technical hexachlorocyclohexane usage and its
contamination consequences in the environment: From 1948 to
1997. Sci. Total EnViron. 1999, 232, 121–158.
(3) Vijgen, J. The legacy of lindane HCH isomer production. A Global
Overview of Residue Management, Formulation and Disposal,
Main Report. International HCH and Pesticides Association
(IHPA), 2006.
(4) Pal, R.; Bala, S.; Dadhwal, M.; Kumar, M.; Dhingra, G.; Prakash,
O.; Prabagaran, S. R.; Shivaji, S.; Cullum, J.; Holliger, C.; Lal,
R. Hexachlorocyclohexane-degrading bacterial strains Sphingomo-
nas paucimobilis B90A, UT26 and Sp+, having similar lin genes,
represent three distinct species, Sphingobium indicum sp. nov.,
Sphingobium japonicum sp. nov. and Sphingobium francense sp.
nov., and reclassification of [Sphingomonas] chungbukensis as
Sphingobium chungbukense comb. nov. Int. J. Syst. EVol. Micro-
biol. 2005, 55, 1965–1972.
(5) Nagata, Y.; Endo, R.; Ito, M.; Ohtsubo, Y.; Tsuda, M. Aerobic
degradation of lindane (γ-hexachlorocyclohexane) in bacteria and
its biochemical and molecular basis. Appl. Microbiol. Biotechnol.
2007, 76, 741–752.
(6) Nagata, Y.; Prokop, Z.; Sato, Y.; Jerabek, P.; Kumar, A.; Ohtsubo,
Y.; Tsuda, M.; Damborsky, J. Degradation of ꢀ-hexachlorocy-
clohexane by haloalkane dehalogenase LinB from Sphingomonas
paucimobilis UT26. Appl. EnViron. Microbiol. 2005, 71, 2183–
2185.
(7) Sharma, P.; Raina, V.; Kumari, R.; Malhotra, S.; Dogra, C.;
Kumari, H.; Kohler, H.-P. E.; Buser, H. R.; Holliger, C.; Lal, R.
Haloalkane dehalogenase LinB is responsible for ꢀ- and δ-hexachlo-
rocyclohexane transformation in Sphingobium indicum B90A.
Appl. EnViron. Microbiol. 2006, 72, 5720–5727.
(8) Wu, J.; Hong, Q.; Han, P.; He, J.; Li, S. A gene linB2 responsible
for the conversion of ꢀ-HCH and 2,3,4,5,6-pentachlorocyclohex-

New Metabolites in the Degradation of R- and γ-HCH
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6603
anol in Sphingomonas sp. BHC-A. Appl. Microbiol. Biotechnol.
2006, 73, 1097–1105.
(15) Mu¨nster, J.; Hermann, R. S.; Koransky, W.; Hoyer, G. A. On the
role of pentachlorocyclohexene in the metabolism and action of
hexachlorocyclohexane. I. Synthesis of ꢀ-pentachlorocyclohexene
and its identification as the monodehydrochlorination product of
R-hexachlorocyclohexane. Hoppe-Seyler’s Z. Physiol. Chem. 1975,
356, 437–447.
(16) Suar, M.; Hauser, A.; Poiger, T.; Buser, H. R.; Muller, M. D.;
Dogra, C.; Raina, V.; Holliger, C.; van der Meer, J. R.; Lal, R.;
Kohler, H.-P. E. Enantioselective transformation of R-hexachlo-
rocyclohexane by the dehydrochlorinases LinA1 and LinA2 from
the soil bacterium Sphingomonas paucimobilis B90A. Appl.
EnViron. Microbiol. 2005, 71, 8514–8518.
(9) Ito, M.; Prokop, Z.; Klvana, M.; Ohtsubo, Y.; Tsuda, M.;
Damborsky, J.; Nagata, Y. Degradation of ꢀ-hexachlorocyclo-
hexane by haloalkane dehalogenase LinB from γ-hexachlorocy-
clohexane-utilizing bacterium Sphingobium sp. MII1205. Arch.
Microbiol. 2007, 188, 313–325.
(10) Raina, V.; Hauser, A.; Buser, H. R.; Rentsch, D.; Sharma, P.;
Lal, R.; Holliger, C.; Poiger, T.; Mu¨ller, M. D.; Kohler, H.-P. E.
Hydroxylated metabolites of ꢀ- and δ-hexachlorohexane: Bacterial
formation, stereochemical configuration, and occurence in ground-
water at a former production site. EnViron. Sci. Technol. 2007,
41, 4292–4298.
(11) Wu, J.; Hong, Q.; Sun, Y.; Hong, Y.; Yan, Q.; Li, S. Analysis of
the role of LinA and LinB in biodegradation of δ-hexachlorocy-
clohexane. EnViron. Microbiol. 2007, 9, 2331–2340.
(12) Prokop, Z.; Monincova, M.; Chaloupkova, R.; Klvana, M.; Nagata,
Y.; Janssen, D. B.; Damborsky, J. Catalytic mechanism of the
haloalkane dehalogenase LinB from Sphingomonas paucimobilis
UT26. J. Biol. Chem. 2003, 278, 45094–45100.
(17) Nagasawa, S.; Kikuchi, R.; Nagata, Y.; Takagi, M.; Matsuo, M.
Aerobic mineralization of γ-HCH by Pseudomonas paucimobilis
UT26. Chemosphere 1993, 26, 1719–1728.
(18) Nagasawa, S.; Kikuchi, R.; Matsuo, M. Indirect identification of
an unstable intermediate in γ-HCH degradation by Pseudomonas
paucimobilis UT26. Chemosphere 1993, 26, 2279–2288.
(13) Kmunicek, J.; Hynkova, K.; Jedlicka, T.; Nagata, Y.; Negri, A.;
Gago, F.; Wade, R. C.; Damborsky, J. Quantitative analysis of
substrate specificity of haloalkane dehalogenase LinB from
Sphingomonas paucimobilis UT26. Biochemistry 2005, 44, 3390–
3401.
(14) Trantirek, L.; Hynkova, K.; Nagata, Y.; Murzin, A.; Ansorgova,
A.; Sklenar, V.; Damborsky, J. Reaction mechanism and stereo-
chemistry of γ-hexachlorocyclohexane dehydrochlorinase LinA.
J. Biol. Chem. 2001, 276, 7734–7740.
Received for review February 14, 2008. Revised manuscript received
May 7, 2008. Accepted May 7, 2008. This work was supported by grants
under the Indo Swiss Collaboration in Biotechnology (ISCB) from the
Swiss Agency for Development and Co-operation (SDC) (Berne,
Switzerland) and from the Department of Biotechnology (DBT)
(Ministry of Science and Technology, Government of India).
JF800465Q