Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

Article abstract of DOI:10.1016/j.febslet.2014.02.056

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.

Full text of DOI:10.1016/j.febslet.2014.02.056

FEBS Letters 588 (2014) 1616–1622
journal homepage: www.FEBSLetters.org
Biochemical and structural characterisation of a haloalkane
dehalogenase from a marine Rhodobacteraceae
Halina R. Novak a, Christopher Sayer a, Michail N. Isupov a, Dorothee Gotz b, Andrew Mearns Spragg b,
Jennifer A. Littlechild a,
⇑
a The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
b Aquapharm Biodiscovery Ltd., European Centre for Marine Biotechnology, Dunstaffnage, Oban, Argyll PA37 1QA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subse-
quently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards
the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The
crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic
active site cavity. A deeper substrate binding pocket defines the enzyme preference towards sub-
strates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind
the distal halogen group of extended di-halogenated substrates.
Received 7 February 2014
Revised 19 February 2014
Accepted 21 February 2014
Available online 5 March 2014
Edited by Stuart Ferguson
Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Keywords:
Haloalkane dehalogenase
Marine Rhodobacteraceae
Three-dimensional structure
Catalytic activity
1. Introduction
belongs to the a/b hydrolase protein fold family with the active site
located on the interface of the core and the cap domain, the latter
determining substrate specificity. More recently, the first eukary-
otic HLD from a purple sea urchin has been biochemically charac-
terised [12].
Haloalkane dehalogenases (HLDs) (EC 3.8.1.5) catalyse the
conversion of halogenated alkanes into their corresponding alcohol
products and hydrogen halides. They have potential applications in
biocatalysis, biosensors and cell imaging, as well as in the bioreme-
diation of recalcitrant and carcinogenic halogenated by-products
from organic synthetic reactions and halogenated pesticides and
insecticides [1,2].
The HLDs from the bacteria Xanthobacter autotrophicus (DhlA)
[3,4], Rhodococcus rhodochrous (DhaA) [5], Sphingomonas paucimo-
bilis (LinB) [6,7], Bradyrhizobium japonicum USDA110 (DbjA) [8,9],
Mycobacterium tuberculosis (DmbC) [10] and Plesiocystis pacifica
SIR-1 (DppA) [11] have been biochemically and structurally
characterised and are two domain proteins. The core domain
The HLD enzymatic reaction proceeds via a SN2 type nucleo-
philic substitution mechanism [1]. The first step is the nucleophilic
attack by the carboxyl group of the conserved aspartate on the car-
bon atom attached to the halogen of the substrate. This produces
an ester intermediate complex where the carboxyl oxygen of the
catalytic aspartic acid replaces the halogen. The second step of
the reaction is the nucleophilic attack on the carboxyl group of
the ester intermediate by a water molecule to release the alcohol
product. For the reaction to proceed, the catalytic aspartate oxygen
which is not involved in the ester intermediate adduct has to be
positioned in the oxyanion hole formed by the main chain nitro-
gens. The catalytic water is activated by a histidine/carboxylic acid
dyad.
Abbreviations: HLD, haloalkane dehalogenase; L-HAD, L-Haloacid dehalogenase;
DhlA, Xanthobacter autotrophicus haloalkane dehalogenase; DhaA, Rhodococcus
rhodochrous haloalkane dehalogenase; DmbC, Mycobacterium tuberculosis haloal-
kane dehalogenase; LinB, Sphingomonas paucimobilis haloalkane dehalogenase;
DbjA, Bradyrhizobium japonicum USDA110 haloalkane dehalogenase; DppA, Plesio-
cystis pacifica SIR-1 haloalkane dehalogenase; DspA, Stronglocentrotus purouratus
haloalkane dehalogenase; HanR, Rhodobacteraceae family haloalkane dehalogenase;
1HO, 1-hexanol; BAM, benzamidine; PMSF, phenylmethysulfonyl fluoride
Crystal structures of different HLD enzymes have allowed the
identification of the catalytically important residues, which have
been confirmed by site-directed mutagenesis [4,6,13]. A catalytic
pentad has been identified consisting of a catalytic triad of Asp,
His and Asp/Glu and a Trp-Trp or Trp-Asn pair, which stabilises
the halide leaving group [1]. The Trp-Trp or Trp-Asn pair of
residues are essential for activity and are responsible for binding
⇑
Corresponding author.
E-mail address: J.A.Littlechild@exeter.ac.uk (J.A. Littlechild).
http://dx.doi.org/10.1016/j.febslet.2014.02.056
0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

H.R. Novak et al. / FEBS Letters 588 (2014) 1616–1622
1617
the substrate in the correct orientation by stabilizing both the tran-
2.2. Enzyme activity
sition state and the halide ion released during the carbon–carbon
bond break. The division of HLDs into three phylogenetic subfam-
ilies was defined by Chovancova et al. in 2007 [14], depending on
the amino acid content of the catalytic pentad. Classification of
these HLDs into four groups based on substrate specificity revealed
there was no clear correlation between substrate specificity and
phylogenetic subfamilies [15]. More recently, the characterisation
of a HLD from Agrobacterium tumefaciens that has a unique
halide-stabilizing tyrosine residue (Tyr109) in place of the con-
served Trp residue [16] has required an extension of the existing
phylogenetic families.
The HLD activity was measured using a modified colorimetric
assay, based on the method of Holloway et al. [23]. The assay solu-
tion had a final concentration of 1 mM HEPES, 1 mM EDTA, 20 mM
sodium sulfate, 10 mM substrate, 20
initiate the assay, 180 l of dehalogenase assay solution was mixed
with 20 l of purified protein (2 mg/ml). The reaction was followed
lg/ml phenol red, pH 8.2. To
l
l
by measuring a decrease in absorbance at 540 nm over 1 h. To pro-
duce a standard curve, the dehalogenase assay solution was mixed
with HCl to a final concentration between 0 and 2 mM in a total
volume of 200 ll.
Over the last decade the marine environment has been recogni-
sed as a potential source of novel enzymes [17]. The oceans are
known to contain abundant organohalogens, which are thought
to be produced mainly by marine algae. Some species of polychaete
tube worms also produce a wide range of structurally diverse hal-
ogenated compounds [18]. These compounds are often cytotoxic
and are thought to be part of the organism’s defence mechanisms.
Microorganisms living in symbiosis with algae and tube worms
may have evolved detoxification enzymes such as dehalogenases.
In the search of novel dehalogenase enzymes, a marine Rhodob-
acteraceae species was isolated from the surface of a tube worm.
2.3. Crystallization
The purified HanR enzyme was concentrated until a final con-
centration of 10 mg/ml was reached. The concentrated protein
was subjected to microbatch crystallization screens at 18 °C using
an Oryx Robot (Douglas Instruments). The best crystals were
grown from a mixture of equal volumes of protein and precipitant
solution containing 0.15 M MgCl2, 0.1 M Tris–HCl and 15% PEG
4000, pH 8.0. The native crystals were frozen using a cryoprotec-
tant containing 0.1 M Tris–HCl pH 8.0, 0.1 M NaCl, 25% PEG 4000
and 30% PEG 400. The substrate soak was conducted by soaking
the HanR crystal for 1 min in 0.1 M Tris–HCl, 20% PEG 3350, 30%
PEG 400, 25 mM 1-bromohexane, pH 4.0.
The wild-type bacterium tested positive for
L-haloacid dehalogen-
ase ( -HAD) activity. In order to obtain sufficient quantities of this
L
enzyme for biochemical characterisation, the Rhodobacteraceae
genome was partially sequenced so that the gene could be cloned
and the protein over-expressed [19]. In addition to the presence of
2.4. Data collection and structure determination
a novel L-HAD which has been biochemically and structurally char-
acterised [19], a putative HLD was also identified called HanR.
This paper describes the biochemical and structural character-
isation of a HLD enzyme from a marine source which has been
isolated from the surface of a tube worm.
Diffraction data were collected at the Diamond Light Source
synchrotron, UK. Data were processed and scaled with the pro-
grams XDS [24] and Aimless [25] using the Xia2 [26] pipeline. Fur-
ther data analysis and refinement were performed using the CCP4
suite of programs [27].
2. Material and methods
The HanR native structure was solved by molecular replace-
ment (MR) with the program MOLREP [28] using the LinB model
(PDB: 1CV2; [6]). The MR solution was submitted to an automated
refinement procedure using ARP/wARP version 7.0.1 [29]. The
resulting model was manually rebuilt in COOT [30] and refined
with REFMAC5 [31].
The structure of the product bound 1-hexanol (1HO) complex
was solved by MR using the refined structure of the native HanR.
The dictionary definition for the ligand was built using JLIGAND
[32]. The quality of the structures was checked using the program
PROCHECK [33]. The program PyMOL [34] was used to produce
Figs. 2a, 3 and 4.
2.1. Gene identification, cloning and overexpression
A Rhodobacteraceae family bacterium (Rhb) isolated from a
Polychaeta worm was collected from Tralee beach, Argyll, UK.
The Rhb genomic DNA was extracted and the genome was
sequenced using an Illumina GA2 sequencer. As there was low gen-
ome identity to previously sequenced genomes, De novo assembly
was used to arrange the 72 bp paired-end reads into 1082 contigs
using Velvet, version 0.7.63 [20]. A preliminary genome of 3.77
Mbp was assembled into 795 contigs. A putative HLD gene called
HanR was identified using the NCBI BLAST tool [21] on a Galaxy
bioinformatics pipeline [22]. The HanR gene was cloned from geno-
mic DNA into the pET-28a plasmid with a N-terminal His-tag. The
HanR protein was over-expressed in BL21-CodonPlus (DE3) Roset-
ta2 Escherichia coli.
3. Results and discussion
3.1. Biochemical characterisation
The cell pellet was re-suspended in buffer A (0.1 M Tris–HCl,
0.1 M NaCl, 0.5 mM EDTA, 1 mM BAM, 1 mM PMSF, pH 8.2) con-
taining 0.01 M imidazole, lysed by sonication and centrifuged to
remove cell debris. A nickel affinity chromatography column (GE
Healthcare) was equilibrated with buffer A, the cell extract loaded
and the unbound protein washed off with buffer A containing
0.01 M imidazole. The bound protein was eluted in buffer A with
0.5 M imidazole.
The fractions corresponding to the protein were concentrated
using a 10 kDa membrane (Vivaspin 20, Vivascience) at 3000Âg,
at 4 °C until the final volume reached 1 ml. The concentrated pro-
tein sample was further purified on a 120 ml Superdex 75 GF chro-
matography column which was eluted over 1 column volume of
buffer A.
The specific activity of the HanR was measured with a variety of
substrates (Fig. 1). The HanR is active towards both chloroalkanes
and bromoalkanes with preference towards longer chain linear
substrates.
Activity of HanR towards brominated compounds appears to be
higher than towards their chlorinated equivalents, which is not
surprising, as the energy of C–Br bond is lower than that of C–Cl.
This is also in line with the abundance of brominated organic com-
pounds in the marine environment. The DppA HLD from the mar-
ine bacteria Plesiocystis pacifica [11] is not active towards any
chlorinated compounds. Substrate variations between the HLD en-
zymes are thought to arise from differences in the size, shape and
hydrophobicity of the cap domain [14]. The differences in substrate
specificity are difficult to infer from protein sequence only.

1618
H.R. Novak et al. / FEBS Letters 588 (2014) 1616–1622
the native and complex structures were 24.6% and 18.4% (Table 1).
The G-factors calculated for each model confirmed these structures
have acceptable stereochemical properties [33]. Proline residues
37, 216 and 244 are in the cis conformation in both the native
and complex structures.
The approximate dimensions of the HanR monomer are
40 Â 40 Â 50 Å. The HanR adopts the typical
consisting of a core and cap domain (Fig. 2a). The core domain is
conserved in all of the /b hydrolase fold superfamily proteins
and is composed of 8 b-strands surrounded by 6 -helices, two
a/b hydrolase fold
a
a
on one side of the b-sheet and 4 on the other. The b-sheet is of
mixed type with direction + À + + + + + + and topology +1, +2,
À1x, +2x, +1x, +1x, +1x [35]. The cap domain (residues 132–233)
is located between strands 6 and 7 of the core domain b-sheet. It
is built up of 6
a-helices (a3–a8) and is the most variable region
of the HLD enzymes. The overall structure of the HanR is very
Fig. 1. The specific activity of HanR towards halogenated compounds with varying
similar to LinB, DmbC, DhaA and DbjA HLD enzymes with C
RMSDs of less than 1 Å.
a
structures, carbon chain length and different positions and degree of chlorination or
bromination displayed in l .
mol sÀ1 mgÀ1
The asymmetric unit of the native structure contains two
enzyme monomers which appear to form a dimer related by a
molecular dyad, however only a small portion of the surface area
(8.7%) is buried upon its formation. This dimer differs from the
crystallographic dimer in the product complex structure by a rela-
tive rotation of several degrees between the monomers. In the di-
mer observed in the crystal structure the adjacent subunit hinders
the access to the enzyme active site. The estimated size of the pro-
tein in solution corresponds to a monomer, as determined by size
exclusion chromatography. As most related HLDs are reported to
Therefore, in order to elucidate the structural features responsible
for the preference of HanR towards long chain substrates, the crys-
tal structure was determined.
3.2. HanR structure
3.2.1. Overall structure
The native and product complex structures were refined to res-
olutions of 1.7 and 1.6 Å, respectively. The resulting Rfree values for
Table 1
The data processing and refinement statistics for the HanR structures.
Crystal
Native HanR
Complex HanR
I03
Beamline (Diamond)
Resolution (Å)
I24
44.4–1.67 (1.71–1.67)a
47.84–1.61 (1.66–1.61)
a
Wavelength (Å)
0.9778
0.97625
Space group
P21
P21212
Cell dimensions (Å),
No. of protomers in A.U.
Solvent content (%) VM (Å3 DaÀ1
Unique reflections
Redundancy
a, b, c = 55.0 Å, 75.5 Å, 64.9 Å;
a
,
c
= 90° b = 92.9°
a, b, c = 64.5 Å, 71.41 Å, 59.75 Å;
a
, b,
c
= 90°
2
1
a
a
)
40 (2.1)
41.1 (2.1)
61186
3.4 (3.3)
36192
12.8 (12.6)
99.9 (100)
a
a
a
a
Completeness
99.6 (99.6)
a
a
<(I)/
r(I)>
14.2 (2.0)
26.0 (3.6)
a
Rsym (%)
4.3 (51.0)
6.1 (74.7)a
15.3
Overall R-factor (%)
Rfree (5% total data)%
Residues modelled
20.4
24.6
A (T3-L292)
18.7
A (T2-L292)
B (T3-L292)
No. of waters modelled
No of chlorines modelled
No of ligands modelled
RMSD bond length (Å)
RMSD bond angle (°)
Wilson B factor (Å2)
Average B factor
Protein (Å2)
466
2
–
352
1
1
0.007 (0.019)b
1.24 (1.95)b
30.0
0.013 (0.022)b
1.35 (1.95)b
27.4
25.8
34.2
33.2
–
22.8
38.8
16.23
33.71
0.09
Water (Å2)
Chlorine (Å2)
Ligand (Å2)
REFMAC RMS error estimate (Å2)
0.126
Ramachandran analysis (% of residues)
Most favoured
Additionally allowed
Generously allowed
Disallowed
88.9
10.5
0.6
0
91.2
8.4
0.4
0
G-factor
0.1
0
a
Values for the outer resolution shell are given in brackets.
P P
P P
P
P
b
Target values are given in parentheses. Rsym
=
J|<Ih> À IJ(h)|/
JI(h), where I(h) is the intensity of the reflections h, h is the sum over all the reflections and J is
h
h
P
P
the sum over J measurements of the reflections. Rcryst
calculation were performed by PROCHECK [33].
=
||Fo|-|Fc||/ |Fo|. Wilson B-factor was estimated by SFCHECK [39]. The Ramachandran plot analysis and G-factor

H.R. Novak et al. / FEBS Letters 588 (2014) 1616–1622
1619
be monomers, the dimer is most likely to be an artefact of
crystallisation.
of residues. Currently four different pentads [14,16] have been
identified from the various HLD enzymes characterised to date.
HanR and LinB (64% amino acid sequence identical) both belong
to the HLD-II subfamily. The catalytic triad (Fig. 2b) consists of
3.3. Catalytic pentad
Asp106 (located between b-strand 5 and
a-helix 2), His271 (lo-
The active sites of Han enzymes are characterised by a catalytic
pentad [14]. This consists of a catalytic triad and a stabilizing pair
cated between b-strand 8 and 310 helix 9) and Glu130 (located
within b-strand 6). The side chains of Trp107 and Asn36 bind the
Fig. 2. (A) Folding of the HanR monomer presented as a cartoon diagram. The catalytic Asp106 residue is displayed as a grey CPK model and
loops are coloured in turquoise, magenta and pink respectively. (B) The sequence alignment of HanR and LinB. The secondary structure of HanR is shown above the sequence
with a spiral and an arrow representing -helices and b-strands respectively using ESPript [40]. The numbering of -helices and b-strands is consistent with Fig. 2a.
a-helices (H), b-strands (S) and
a
a
Conserved residues are shown in red boxes and residues with similar properties are in blue boxes. The HLD catalytic pentad is highlighted with stars in cyan beneath the
sequence alignment.

1620
H.R. Novak et al. / FEBS Letters 588 (2014) 1616–1622
substrate halogen, while their main chain nitrogen atoms form the
oxyanion hole.
As the crystallisation media contained chloride ions, the halide
binding site of HanR contains a bound chloride ion at full occu-
pancy. The catalytic pentad residues in HanR and the position of
the chloride ion match those of LinB [36] very closely (Fig. 3).
active site resulting in a larger substrate binding pocket. This
observation is confirmed by the well-defined electron density
and low B-factors of the Arg145 residue in both native and com-
plex structures.
The product 1-butanol (1BO) in the LinB complex structure
binds in a similar conformation to the 1HO in the active site of
the HanR product complex but in HanR it is located deeper into
the active site. The disorder of the 1HO product in the HanR active
site is thought to be due to the wider substrate channel which can
also bind cyclic haloalkanes.
3.4. Product binding
To map the HanR substrate pocket, crystals were soaked with
1-bromohexane. The electron density of the HanR 1-bromohexane
substrate soaked crystal revealed a fully dehalogenated 1HO prod-
uct in the active site. This was modelled in two conformations,
each having a partial occupancy of 0.5 (Fig. 4). The 1HO molecule
sits in a hydrophobic pocket formed by residues Leu247, Val210,
Phe150 and Phe142.
There is little structural difference between the native and
product complex structures (RMSD of 0.35 Å). The presence of
the 1HO product shifts Arg145 2.7 Å away from the hydrophobic
3.5. Structural features responsible for substrate specificity
For HLD catalysis, the enzyme halide site should bind the sub-
strate halogen, while the carbon attached to the halogen and the
adjacent carbon should have a fixed position in the active site. To
achieve this, HLDs need to bind the substrate tightly in an ex-
tended conformation. DhlA, a HLD with a small active site displays
high, activity towards 1,2-dichloroethane while the enzymes with
Fig. 3. A superimposition of the active site residues in the HanR (pink) and the LinB (grey), with the chlorine ion in the active site cavity shown as a green sphere. The
significantly different residues between the active site of these HLDs are highlighted in magenta in the HanR structure.
Fig. 4. A stereo diagram showing the active site cavity of the HanR with the 2Fo–Fc electron density map contoured at 1 r. The 1HO product modelled in two conformations is
displayed as black sticks. The chloride ion is shown as a green sphere. Active site residues are shown as sticks.

H.R. Novak et al. / FEBS Letters 588 (2014) 1616–1622
1621
larger active site pockets (HanR, LinB) are inert towards this
substrate.
The HLD active site is built up of an entrance tunnel and a
hydrophobic pocket for substrate binding [37]. The residues lining
the substrate pocket mainly belong to the cap domain and define
substrate specificity. Minor differences in the substrate pocket
can dramatically affect the substrate specificity of very similar
HLDs [15].
4.1. Accession number
The HanR gene and its protein sequence have been deposited in
Genbank: KF032932. Structure factors and coordinates of the final
models have been deposited in the PDB: 4BRZ and 4C6H.
Acknowledgements
We observe a difference in substrate specificity between the
highly similar HLD-II family enzymes, HanR and LinB [15]. LinB
displays a significant drop of activity as the carbon chain becomes
longer from the substrates 1-bromobutane to 1-bromoxehane. This
would infer a lower activity towards the longer 1-bromooctane
substrate, which HanR prefers to 1-bromoxehane. There are signif-
icant differences between the entrance tunnels of these two
enzymes (Fig. 3), where in HanR, Ser176 replaces Leu177 of LinB,
and on the opposite side of the entrance tunnel Arg145-Ala146 re-
places the Gln146-Asp147 pair of residues in LinB. Ile211 which is
located close to the halide binding site in LinB is replaced by the
smaller Val210 in HanR. Additionally, some conserved residues be-
tween the two enzymes such as Phe150, Leu247, and Phe142 in
HanR have different conformations or are shifted from the posi-
tions of their equivalents in LinB, thereby increasing the volume
of the active site cavity in HanR. Although these changes may con-
tribute to the different substrate specificity between these two
enzymes, we propose that the differences in the substrate binding
pocket are more likely to account for the substrate variations
observed.
We thank Diamond Light Source for access to beamlines I03 and
I24 (proposal numbers mx2032 and mx6851) and the beamline
scientists. We thank Konrad Paszkiewicz, University of Exeter, for
help with the genome sequencing of the Rhodobacteraceae organ-
ism. The Biotechnology and Biological Science Research Council,
UK and Aquapharm Biodiscovery, Oban are acknowledged for
PhD studentship funding (HRN). C.S. acknowledges a PhD GTA bur-
sary from the University of Exeter. M.I. is grateful to the University
of Exeter and a BBSRC funded ERA-IB Grant BB/L002035/1. Funding
in J.A.L.s laboratory has been supported by the Wellcome Trust, EU,
BBSRC and EPSRC.
References
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The importance of the extended or flexible substrate channel for
activity towards long chain substrates was demonstrated for the
DhlA enzyme [38]. Specific activity of the wild type DhlA towards
1,6 dibromohexane was only 0.073 l
mol minÀ1 mgÀ1. This was
increased threefold in the F164A mutant DhlA enzyme by freeing
the space occupied by the phenyl ring. Interestingly, the D170A
surface mutation of DhlA, which disrupted the salt bridge holding
a loop in place, showed a twofold higher specific activity towards
1,6 dibromohexane, probably due to the increased flexibility of
the substrate channel.
The most significant differences between HanR and LinB are at
the bottom of the substrate pocket where Ile138, Met253 and
Leu248 of LinB form a hydrophobic cluster, limiting the size of
the bound substrate. Substitutions of Ile138 (LinB) by Arg136
(HanR) and Met253 by the smaller Val252, in addition to a differ-
ent conformation of the conserved Leu247, significantly expand
the space in the substrate channel of HanR to accommodate longer
chain substrates in comparison to LinB. Positively charged Arg136
in HanR at the bottom of the active site is not observed in other
HLDs. This will facilitate the binding of the distal halogen group,
as demonstrated by the high activity of HanR towards 1,6 dichloro-
Crystallization and preliminary crystallographic analysis of
a haloalkane
dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110. Acta
Crystallogr. F 63, 294–296.
[9] Sfetsas, C.C., Milios, L., Skopelitou, K., Venieraki, A., Todou, R., Flemetakis, E.,
Katinakis, P. and Labrou, N.E. (2009) Characterization of 1,2-dibromoethane-
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