Báo cáo khoa học: Role of Acinetobacter calcoaceticus 3,4-dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids Kohsuke Honda, Michihiko Kataoka, Eiji Sakuradani and Sakayu Shimizu doc

Role of
Acinetobacter calcoaceticus
3,4-dihydrocoumarin hydrolase
in oxidative stress defence against peroxoacids
Kohsuke Honda, Michihiko Kataoka, Eiji Sakuradani and Sakayu Shimizu
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan
The physiological role of a bifunctional enzyme,
3,4-dihydrocoumarin hydrolase (DCH), which is capable of
both hydrolysis of ester bonds and organic acid-assisted
bromination of organic compounds, was investigated.
Purified DCH from Acinetobacter calcoaceticus F46 cata-
lysed dose- and time-dependent degradation of peracetic
acid. The gene (dch) was cloned from the chromosomal
DNA of the bacterium. The dch ORF was 831 bp long,
corresponding to a protein of 272 amino acid residues, and
the deduced amino acid sequence showed high similarity to
those of bacterial serine esterases and perhydrolases. The dch
gene was disrupted by homologous recombination on the
A. calcoaceticus genome. The dch disruptant strain was
more sensitive to growth inhibition by peracetic acid than the
parent strain. On the other hand, the recombinant Escheri-
chia coli cells expressing dch were more resistant to peracetic
acid. A putative catalase gene was found immediately
downstream of dch, and Northern blot hybridization ana-
lysis revealed that they are transcribed as part of a polycis-
tronic mRNA. These results suggested that in vivo DCH
detoxifies peroxoacids in conjunction with the catalase, i.e.
peroxoacids are first hydrolysed to the corresponding acids
and hydrogen peroxide by DCH, and then the resulting
hydrogen peroxide is degraded by the catalase.
Keywords: Acinetobacter calcoaceticus; catalase; 3,4-dihydro-

coumarin hydrolase; perhydrolase; peroxoacid.
In a previous study, we found and isolated a novel
lactonohydrolase, 3,4-dihydrocoumarin hydrolase (DCH),
from Acinetobacter calcoaceticus F46 [1]. The amino acid
sequences of the N-terminal and internal peptide of DCH
exhibited high similarity to those of several serine-hydro-
lases and perhydrolases (nonhaem haloperoxidases). Fur-
thermore, the enzyme also showed both hydrolysis activity
toward aromatic lactones, such as 3,4-dihydrocoumarin
(Scheme 1), and organic acid-assisted bromination activity
toward monochlorodimedon (2-chloro-5,5-dimethyl-1,3-
cyclohexanedione).
Perhydrolases were originally identified as the enzymes
catalysing halogenation reactions of various organic com-
pounds, because these enzymes catalyse the halogenation of
various organic compounds in the presence of hydrogen
peroxide, halide ions, and organic acids. Various perhydro-
lases have been isolated, mainly from Pseudomonas and
Streptomyces species [2–8]. Several of these species are
known to produce halogenated metabolites. Perhydrolases
had been, as a result of this, thought to be involved in the
synthesis of halogenated metabolites in vivo, and had been
called Ônonhaem haloperoxidasesÕ, to distinguish them from
the ÔtrueÕ haloperoxidases, which are haem- or vanadium-
dependent enzymes [9]. However, there is now evidence that
perhydrolases do not participate in the biosynthesis of
halogenated compounds. A perhydrolase-deficient mutant
of Pseudomonas fluorsecens yielded a chlorinated metabo-
lite, pyrrolnitrin, like the parent strain [4], and the Strep-
tomyces enzyme, which chlorinates tetracycline, is not

related to the perhydrolase [10]. X-ray crystallographic
analysis of the perhydrolase of Streptomyces aureofaciens
revealed an a/b-hydrolase fold, with a Ser-Asp-His catalytic
triad [11] that is conserved in the serine-hydrolase family.
Interestingly, this catalytic triad is also highly conserved in
the amino acid sequences of perhydrolase [12,13]. Based on
recent results, a reaction mechanism has been proposed for
the halogenation reaction catalysed by a perhydrolase with
peroxoacid and hypohalous acid as reaction intermediates.
The enzyme catalyses the peroxidation of the organic acid to
O
O
H
2
O
OH
COOH
3
,4-Dihydrocoumarin
3-(2-Hydroxyphenyl)propionic aci
d
Scheme 1.
Correspondence to M. Kataoka, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University,
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606–8502,
Japan. Fax/Tel.: +81 75 7536462,
E-mail:
Abbreviations: DCH, 3,4-dihydrocoumarin hydrolase; dch,gene
encoding 3,4-dihydrocoumarin hydrolase; cat, gene encoding putative
catalase; IPTG, isopropyl thio-b-

D
-galactoside; LB, Luria–Bertani.
Enzymes: 3,4-dihydrocoumarin hydrolase (EC 3.1.1.35); catalase
(EC1.11.1.6); chloroperoxidase (EC1.11.1.10).
Note: The nucleotide sequences reported here have been submitted
to DDBJ/EMBL/GenBank databases under accession number
AB092339.
Note: a web site is available at />lab-e/index-e.html
(Received 30 September 2002, revised 22 November 2002,
accepted 28 November 2002)
Eur. J. Biochem. 270, 486–494 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03407.x
peroxoacid in the presence of H
2
O
2
through a reaction
mechanism that closely resembles transesterification. Sub-
sequently, the halide ion is oxidized by the peroxoacid to
hypohalous acid, and then nonenzymatic halogenation of
organic compounds such as monochlorodimedon by the
resulting hypohalous acid takes place [1,14–16] (Fig. 1).
Actually, DCH also requires organic acids, such as formic
acid, acetic acid, propionic acid and n-butyric acid, to
catalyse the halogenation reaction [1]. In addition, it has
been shown that several lipases catalyse the synthesis of
peroxoacids [17]. These results suggest that the halogenation
reactions catalysed by perhydrolase do not reflect the
physiological roles of these enzymes. However, it seems
improbable that perhydrolase in vivo catalyses the formation
of peroxoacids, which are very toxic for cells. So, we

predicted that these enzymes catalyse the reverse reaction,
i.e. hydrolytic degradation of peroxoacids, in vivo.Inorder
to confirm this assumption, we carried out functional
analysis of a bifunctional hydrolase-perhydrolase, with
DCH as a model enzyme. Here we describe the cloning of
the DCH gene (dch)fromA. calcoaceticus F46, a gene
disruption experiment and the physiological role of the
enzyme.
Materials and methods
Chemicals and enzymes
A stock solution of peracetic acid of 32% (w/v) concentra-
tion in equilibrium with < 6% hydrogen peroxide and
40–45% acetic acid (Aldrich Chemical Co.) was used.
Restriction enzymes, alkaline phosphatase (calf intestine),
T4 DNA ligase and Ex Taq DNA polymerase were from
Takara Shuzo Co. (Kyoto, Japan). All other reagents were
commercially available and of analytical grade.
Strains, plasmids and growth media
A. calcoaceticus F46 was previously isolated from a soil
sample [1]. Escherichia coli JM109 was used for gene cloning
and expression. Microorganisms were cultured on Luria–
Bertani (LB) medium [18] at 28 °CforA. calcoaceticus,and
37 °CforE. coli. In selective media, ampicillin and kana-
mycin were used at 100 lgÆmL
)1
and 50 lgÆmL
)1
, respect-
ively. pT7Blue (Novagen) was used for subcloning of the
PCR products. pBluescript II SK(+) (Toyobo, Osaka,

Japan) was used as a cloning vector. pKK223-3 (Amersham
Bioscience) was used as expression vector, and pKT231 [19]
was used for construction of the dch disruptant.
Enzyme assay, SDS/PAGE and protein determination
Acinetobacter strains and the recombinant E. coli strains
were cultured in 3 mL LB medium. For cultivation of the
recombinant E. coli strains, isopropyl thio-b-
D
-galactoside
(IPTG) was added to a final concentration of 2 m
M
.After
cultivation for 12 h, cells were harvested by centrifugation,
resuspended in 1 mL 20 m
M
potassium phosphate buffer
pH 7.0, and disrupted by sonication. The supernatant
obtained on centrifugation (14 000 g,4°C, 20 min) was
used as the enzyme solution.
3,4-Dihydrocoumarin-hydrolysing activity and mono-
chlorodimedon-brominating activity were determined as
described previously [1]. Catalase activity was determined
according to the method of Hildebraunt and Roots [20].
Peracetic acid-hydrolysing activity was measured as des-
cribed below.
SDS/PAGE was performed in a 12.5% polyacrylamide
slab gel using the Tris/glycine buffer system described by
King and Laemmli [21].
Protein concentrations were determined as described by
Bradford [22] using BSA as standard.

Determination of peracetic acid-hydrolysing activity
of purified DCH
DCH was purified from A. calcoaceticus F46 as described
previously [1]. The assay mixture for peracetic acid-hydro-
lysing activity comprised 100 m
M
sodium citrate buffer
pH 5.5, 0.01% (w/v; 1.31 m
M
) peracetic acid and enzyme in
a final volume of 500 lL. Aftera 10 min incubation at 30 °C,
the reaction was stopped by adding 500 lL1
M
HCl. The
amount of peracetic acid remaining in the reaction mixture
was determined as monochlorodimedon-brominating ability
[16]. One-hundred lL of the reaction mixture was added to a
detection mixture comprising 100 m
M
KBr and 50 l
M
monochlorodimedon in a final volume of 2.5 mL. After
10 min at room temperature, the absorbance at 290 nm was
measured with a Shimadzu UV-240 spectrophotometer
(Kyoto, Japan). Blanks without the enzyme and without
peracetic acid were included if necessary. A molar extinction
coefficient of 19 900Æ
M
)1
Æcm

)1
for monochlorodimedon was
used for calculation of enzyme activity. One unit of enzyme
was defined as the amount of enzyme catalysing the
consumption of 1 lmol peracetic acid per min at 30 °C.
Enzyme
Ser
OOC-R
Enzyme
Ser
OH
R-COOHR-COOOH
H
2
O
2
H
+
, Br
-
HBrO H
2
O
H
2
O
Cl
HH
3
C

H
3
C
O
O
Cl
Br
H
3
C
H
3
C
O
O
Monochlorodimedon
Monochloromonobromodimedon
ii
iii
iv
i
Fig. 1. Predicted reaction mechanism of bromination reactions with
bacterial nonhaem haloperoxidases. (i) Nucleophilic attack of an active
serine residue on the carboxyl carbon atom of the organic acid and
formation of an acyl-enzyme intermediate; (ii) hydrolytic cleavage of
the acyl-enzyme on nucleophilic attack by hydrogen peroxide;
(iii) nonenzymatic formation of hypohalous acid from the peroxoacid
and a halide ion; (iv) nonenzymatic halogenation of organic com-
pounds such as monochlorodimedon by the hypohalous acid.
Ó FEBS 2003 Role of 3,4-dihydrocoumarin hydrolase of Acinetobacter (Eur. J. Biochem. 270) 487

Oxidative stress sensitivity assays
To test the susceptibility of A. calcoaceticus strains and the
recombinant E. coli strains to oxidative stress agents, disk
inhibition assays were performed. Cells grown overnight in
2 mL LB medium were mixed with 10 mL LB medium
containing 1% agar at 42 °C and then poured onto an LB
agar plate (135 · 95 mm). For the recombinant E. coli
strains, IPTG was added to a final concentration of 2 m
M
.
Sterilized filter disks containing 10 lL peracetic acid, H
2
O
2
,
or acetic acid at an appropriate concentration, were placed
on the agar plates. The plates were incubated overnight at
28 °CforA. calcoaceticus strains or at 37 °CforE. coli
strains, and then the halos of growth inhibition were
measured.
DNA isolation
Total DNA of A. calcoaceticus waspreparedasfollows.
A. calcoaceticus was cultivated overnight in 500 mL LB
medium. Cells were collected by centrifugation and resus-
pended in 12 mL buffer comprising 25% sucrose, 50 m
M
Tris/HCl pH 8.0, 50 m
M
EDTA. Lysozyme (Seikagaku
Co., Tokyo, Japan) and proteinase K (Wako Pure Chemi-

cals, Osaka, Japan) were added to the mixture at final
concentrations of 2 mgÆmL
)1
and 1.25 mgÆmL
)1
, respect-
ively. After incubation at 37 °C for 30 min, an equal volume
of 2% SDS was added for cell lysis. DNA was purified from
the lysate by phenol/chloroform (1 : 1, v/v) extraction,
precipitation by isopropanol, RNase-treatment, and then
re-precipitation with ethanol. The resultant precipitate, i.e.
total DNA, was dissolved in TE buffer consisting of 10 m
M
Tris/HCl pH 8.0, 1 m
M
EDTA.
Cloning of
dch
from the genome of
A. calcoaceticus
F46
To amplify the fragment of DNA encoding DCH from the
total genomic DNA of A. calcoaceticus F46byPCR,two
highly degenerate primers were designed based on the
amino acid sequences of the N-terminal (VDIFYKDW)
and internal peptide (EDDQVVPFE) of the purified DCH
[1]. The primers used were a sense primer, GTIGA
(C/T)AT(A/T/C)TT(C/T)TA(C/T)AA(A/G)GA(C/T)TGG,
and an antisense primer, TC(A/G)AAIGGIACIAC(C/
T)TG(A/G)TC(A/G)TC(C/T)TC, where ÔIÕ denotes inosine.

The reaction mixture comprised 1 lg total DNA, 250 pmol
each primers, 8 nmol each dNTP and 1.5 U Ex Taq
polymerase in a final volume of 50 lL. The reaction mixture
was run on a thermal cycler (T Gradient; Biometra,
Go
¨
ttingen, Germany) using a program of 1 min at 94 °C,
1minat55°C, and 1 min at 72 °C for 30 cycles. The PCR
product was approximately 0.7 kb in length, and was used
as a probe. Southern blotting and colony hybridization were
performed essentially as described by Sambrook et al.[18]
using an AlkPhos Direct system (Amersham Bioscience).
The probe hybridized to a 5.7-kb XbaIfragmentoftotal
DNA from A. calcoaceticus F46. The enriched gene library
was prepared by separating XbaI-digested total DNA on an
agarose gel, ligating the isolated DNA in the range of 5.7 kb
into pBluescript SK II (+), and then transforming the
ligation mixture into E. coli JM109. Colony hybridization
led to the identification of a single recombinant clone
harbouring the plasmid, which contained the 5.7 kb XbaI
fragment, and the plasmid was named pADH10. A
restriction map of pADH10 is shown in Fig. 2.
Nucleotide sequencing
Nucleotide sequencing was performed by the dideoxy chain
termination method with a CEQ DTCS kit and a CEQ
2000XL DNA analysis system (Beckman Coulter). The
sequencing reaction was performed as described in the
instruction manual.
Expression of
dch

in
E. coli
The dch ORF was amplified by PCR using pADH10 and
the following two oligonucleotides as a template and as
primers, respectively. The sense primer (CAGGAGA
ATTCAAAATGGG) contained an EcoRI recognition site
(in italics), and the antisense primer (GCCTAAGCTTAAA
ACTTAAC) contained a HindIII recognition site (in italics).
The conditions for PCR were the same as those described
above. The PCR-amplified products were ligated into
pT7Blue (designated pDCH1), and sequenced to verify that
they had correctly encoded DCH. A plasmid, designated
pDCH21, was constructed by ligation of the 860-bp
fragment (derived from pDCH1 on EcoRI–HindIII diges-
tion) with pKK223-3, and was then transformed into E. coli
JM109.
Northern hybridization
A. calcoaceticus F46 was grown in LB medium to the early
exponential growth phase. After cells had been harvested by
centrifugation, total RNA was obtained by the acid/
guanidinium thiocyanate/phenol/chloroform method using
ISOGEN (Nippon Gene Co., Tokyo, Japan). Total RNA
was electrophoresed on a 1% agarose gel in 20 m
M
Mops
buffer containing 1 m
M
EDTA and 2.2
M
formaldehyde,

and then transferred to a Hybond-N membrane (Amer-
sham Bioscience) in 20 · NaCl/Cit (1 · NaCl/Cit is 0.15
M
NaCl, 15 m
M
sodium citrate). Internal fragments of dch
(derived from pDCH21 on EcoRI–HindIII digestion) and a
dch cat
unknown
protein
putative
transposase
1 kb
XbaI
XbaI
BglII
EcoT22I
Eco
T22I
5.7 kb
Fig. 2. Proposed gene organization and restriction map of the cloned
region from A. calcoaceticus F46 bearing dch. Proteins encoded by
ORFs other than that of dch are based on their similarity to other
proteins in the protein sequence database (see text for details). Arrows
indicate the direction and extent of sequence determination.
488 K. Honda et al. (Eur. J. Biochem. 270) Ó FEBS 2003
putative catalase gene (derived from pADH10 on EcoT22I
digestion, Fig. 2) were used as specific probes. Hybridiza-
tion was carried out using the AlkPhos Direct system
according to the manufacturer’s protocol. rRNA was used

as a standard for loaded total RNA and was visualized by
ethidium bromide staining.
Construction of a
dch
disruptant
The construction of a gene replacement vector is illustrated
in Fig. 3A. A 913-bp PCR product containing a C-terminal
truncated DHase gene was generated by PCR with primers
dch-I (GAGCTGAATTCTTTGGTCA) containing an
EcoRI site (in italics), and dch-II (ATGTAAGATCTGA
ACTGGACTTCG) containing a BglII site (in italics),
cloned into pT7Blue, excised with EcoRI and BglII, and
then replaced at the EcoRI and BglII sites of pADH10. The
resulting plasmid, named pDHEB, contains the gene
encoding DCH lacking the C-terminal region, in which
Asp and His residues involved in the catalytic triad are
contained. The kanamycin resistance gene (Km
R
), including
its own promoter, was amplified by PCR using pKT231 as a
template. The sequences flanking the BamHI site were
as follows: sense primer, GGATCCGGACCAGTTGGT
GATTTT, and antisense primer, GGATCCTTAGAAAA
ACTCATCGAGC (BamHI recognition are shown in
italics). The amplified Km
R
was excised with BamHI and
then inserted into the BglII site of pDHEB (designated
pDHKm
R

). The resulting dch disruption plasmid,
pDHKm
R
, was linearized and then introduced into A. cal-
coaceticus F46 by electroporation.
Transformation method
A. calcoaceticus F46 was transformed by electropolation
using a Gene Pulser II (Bio-Rad Laboratories) under the
following conditions: 0.1-cm cuvette, 400 W,25lF, and
16 kVÆcm
)1
. Competent cells for electroporation were
prepared by the method of Leahy [23]. After the pulse,
1.5 mL of 30 °C-prewarmed LB was added immediately,
followed by incubation at 30 °C for 4 h and then plating.
E. coli was transformed by the method of Hanahan [24].
Results
Peracetic acid hydrolysis catalysed by DCH
Titration with sodium thiosulfate is commonly used to
measure peracetic acid concentration. For exact determia-
tion, the concentration of peracetic acid needs to be
increased and too high a concentration of peroxoacid
inhibits enzyme activity. Therefore we developed another
method for the determination of small amounts of peracetic
acid (see Materials and methods). As described in the
Introduction, monochlorodimedon is converted to mono-
bromomonochlorodimedon in the presence of peracetic
acid and bromine. Based on this principle, the amount of
peracetic acid could be determined by measuring spectro-
photometrically the decrease in monochlorodimedon. The

standard assay with a peracetic acid solution of known
concentration, obtained by dilution of 32% peracetic acid,
revealed that the concentration could be determined exactly
up to 0.1% (13.1 m
M
) (data not shown).
Under the standard assay conditions, the purified DCH
catalysed the degradation of peracetic acid in a dose- and
time-dependent manner (Fig. 4). In contrast, the cell-free
extractofthedchD Acinetobacter strain did not show
peracetic acid-degradation activity (Table 1). These results
confirmed that DCH catalysed the degradation of peracetic
acid.
Lineweaver–Burk treatment of the data yielded an
apparent K
m
for peracetic acid of 0.390 m
M
at pH 5.0.
This value was lower than those for other substrates, such as
3,4-dihydrocoumarin (0.806 m
M
,pH7.0)andDL-b-acetyl-
thioisobutyrate methyl ester (25.9 m
M
,pH7.0)[1].Onthe
other hand, the V
max
for peracetic acid was 12 600 UÆmg
)1

,
which was much higher than those for other substrates.
Nucleotide and deduced amino acid sequences
The nucleotide sequence of the 5.7 kb XbaIregionof
pADH10 was determined (Fig. 2). An ORF, which exten-
ded from position 231 to 1061 and encoded 276 amino acid
residues, was confirmed to be dch by the N-terminal and
internal amino acid sequences of the purified DCH [1]. The
Fig. 3. Gene disruption of dch in A. calco-
aceticu s F46. (A) Construction of gene dis-
ruption vector pDHKm
R
. ÔDÕ and ÔHÕ within
dch of pADH10 represent Asp and His resi-
dues that are involved in the catalytic triad.
The arrows represent the primers used for
PCR to amplify the catalytic triad-defective
dch. (B) Genomic Southern analysis of XbaI-
digested total DNAs (10 lg each) from the
dchD strain (lane 1) and wild-type strain
(lane 2), with the thermostable alkaline phos-
phatase-labeled dch fragment as a probe.
Ó FEBS 2003 Role of 3,4-dihydrocoumarin hydrolase of Acinetobacter (Eur. J. Biochem. 270) 489
deduced amino acid sequence of DCH exhibited high
similarity to those of the esterases of Pseudomonas putida
MR-2068 (65.1% identity in overall amino acid sequence)
[25], P. fluorescens SIK WI (43.3%) [12,26], and P. fluores-
cens DSM 50106 (22.9%) [27], and the nonhaem haloper-
oxidases of P. putida (76.7%) [28], P. pyrrocinia (67.0%)
[29], S. lividans (65.1%) [7], Rhodococcus erythropolis

(64.6%) [8], and S. aureofaciens (46.7%) [30,31] (Fig. 5).
The active amino acid residues of serine-hydrolases, the
so-called catalytic triad, consisting of the consensus motif,
Gly-X-Ser-X-Gly, and Asp and His residues, were highly
conserved among these proteins. Downstream of dch,an
additional three ORFs were identified (Fig. 2). The second
ORF extending from position 1139 to 2659, encoded a
polypeptide of 506 amino acids with a calculated M
r
of
55 909 Da. The deduced amino acid sequence of this
protein exhibited significant similarity to those of the
catalases of P. fluorescens (84% identity in overall amino
acid sequence) [32], Haemophilus influenzae (84%) [33], etc.
The third ORF (from position 2728 to 3288) encoded a
deduced protein of 186 amino acids with a calculated M
r
of
20 922 Da, which showed no significant similarity to any
other proteins in the protein sequence database. The last
ORF (from position 4390 to 5262) encoded a protein of 290
amino acids (M
r
¼ 33 168 Da) and exhibited high similar-
ity to the transposases of Methylomonas aminofaciens (98%
identity) [34], E. coli (98%) [35], etc.
Expression of
dch
in
E. coli

A cell-free extract of E. coli JM109 transformed with
pDCH21 exhibited specific 3,4-dihydrocoumarin-hydro-
lysing activity of 116 UÆmg
)1
and monochlorodimedon-
brominating activity of 14.3 UÆmg
)1
,i.e. 30-fold increases
in comparison with those of A. calcoaceticus F46 (See
Table 1). E. coli JM109 and E. coli JM109 bearing
pKK223-3 did not have enzyme activity. These results
confirm that DCH catalyses both the hydrolysis and
halogenation reactions. SDS/PAGE of a cell-free extract
of E. coli bearing pDCH21 gave a thick band corresponding
to DCH and revealed that the level of DCH produced by
the transformant was very high (Fig. 6).
Gene disruption of
dch
in
A. calcoaceticus
F46
To determine the physiological role of DCH in A. calco-
aceticus F46, the chromosomal dch gene was destroyed by
homologous recombination with disruption vector
pDHKm
R
as described in Materials and methods. The
transformant was selected for the kanamycin resistance
phenotype. The dch gene disruption was confirmed by
Southern analysis (Fig. 3B). The chromosomal DNAs of

both the wild-type strain and the dchD strain were digested
with XbaI. The signals of the wild-type strain and dchD
strain corresponded to sizes of 5.7 kb and 6.3 kb, respect-
ively. This difference is expected to result from insertion of a
single copy of the Km
R
gene.
3,4-Dihydrocoumarin-hydrolysing activity, monochloro-
dimedon-brominating activity, and peracetic acid-degrading
activity were compared between the wild-type strain and
Fig. 4. Dose- and time-dependent decomposition of peracetic acid
catalysedbyDCH.(A) Dose-dependent decomposition of peracetic
acid. Purified DCH (6.67, 13.3 and 26.7 ng) was added to 500 lLof
the standard reaction mixture. The enzyme reactions were carried out
for 5 min at 30 °C. (B) Time-dependent decomposition of peracetic
acid. The enzyme reactions were carried out at 30 °C, for 5, 10, 15, 20
or 30 min, with 3.33 ng purified DCH. After the reactions had been
terminated by the addition of 500 lL1
M
HCl, 100 lLofthereaction
mixture was transferred to the detection mixture to give a final volume
of 2500 lL. The rates of peracetic acid-degradation were determined
by monitoring the decrease in A
290
, and calculated with a molar
absorption coefficient of 19 900
M
)1
Æcm
)1

for monochlorodimedon.
Table 1. Comparison of the enzyme activities of the A. calcoaceticus
dchD strain and parent strain F46. Preparation of cell-free extracts and
determination of each enzyme activity were performed as described in
the text.
Enzyme activity (UÆmg
)1
) F46 dchD
3,4-Dihydrocoumarin-hydrolysing activity 4.00 0
Monochlorodimedon-brominating activity 0.443 0
Peracetic acid-hydrolysing activity 27.3 0
Catalase activity 105 118
490 K. Honda et al. (Eur. J. Biochem. 270) Ó FEBS 2003
dchD strain (Table 1). A cell-free extract of the dchD strain
did not show any enzyme activity. On the other hand,
catalase activity of the cell-free extract of the dchD strain was
almost same as that of the wild-type (Table 1), and it was
assumed that the expression level of the putative catalase
gene (cat), which was encoded immediately downstream of
dch, was not affected by dch disruption.
Northern analysis
To determine the transcription unit of dch,totalRNA
extracted from early exponential growth phase cells of
A. calcoaceticus F46 was probed with specific internal DNA
fragments derived from either dch or the cat gene located
downstream of dch. Transcripts of  0.8 and  2.6 kb that
hybridized to the dch probe were observed, suggesting that
dch was transcribed as both a monocistronic mRNA and
part of a polycistronic mRNA (Fig. 7). In addition, the
presence of mRNA components of  1.5 and  2.6 kb that

hybridized to cat indicated that cat was also transcribed as
both a single cistron and part of polycistronic mRNA.
These finding that dch and cat were transcribed as parts of
the same polycistronic mRNA indicated that both of them
were regulated by the same promoter.
DCH
GYVTTKDG VDIFYKDWGPRDAPV 23
EST-P
SYVTTKDG VQIFYKDWGPRDAPV 23
EST-F
S TFVAKDG TQIYFKDWGS GKP 21
EST-F1
MAVQWL I AAGVLVGAS VVFWGL SAWMTRR I EAAVP GNGR FVEVDGER F HYYE EGK - - GP P 58
BPO-EST MSYVTTKDG VQIFYKDWGPRDAPV 24
CPO-P
PYVTTKDN VEIFYKDWGPKDAQ P 23
CPO-L
GTVTTSDG TNIFYKDWGPRDGLP 23
HPO-R
PFVTASDG TEIFYKDWGS GRP 21
BPO-A1
PICTTRDG VEIFYKDWGQ GRP 21
BPO-A2
P F I TVGQEN S T S I DLYYEDHGT - - GQP 25
DCH
I F F HHGWPL S SDDWDAQML F FLKEG FRVVAHDRRGHGR STQVW- DGHDMDHYADDVAAVV 82
EST-P
I HFHHGWPL SADDWDAQML F FLAHGYRVVAHDRRGHGR S S QVW- DGHDMDHYADDVAAVV 82
EST-F
VL F SHGWLLDADMWEYQMEYL S SRGYR T I AFDRRGFGR S DQPW- TGNDYDT FADD I AQL I 80

EST-F1
LVMI HGLMGS SRNL TYAL SRQLR EHF RV I T LDR PG SGYS TRHKGTAADL PAQARQVAA F I 118
BPO-EST I H F H H G W P L S A D D W D A Q M L F F L G Q G F R V F A H D R R GHGR S SQV S - DGHDMDHYADDVAAVV
83
CPO-P
I VFHHGWPL SGDDWDAQML F FVQKGYRV I AHDRRGHGR SAQV S - DGHDMDHYAADAFAVV 82
CPO-L
VVF HHGWPL SADDWDNQML F F L SHGYRV I AHDRRGHGR SDQ P S - TGHDMDTYAADVAAL T 82
HPO-R
I MFHHGWPL S SDDWDSQL L FLVQRGYRV I AHDRRGHGR SAQVG - HGHDMDHYAADAAAVV 80
BPO-A1
VVF I HGWPLNGDAWQDQLKAVVDAGYRG I AHDRRGHGH S T PVW- DGYD FDT FADDLNDL L 80
BPO-A2
VVL I HGF PL SGH SWERQ SAAL LDAGYRV I TYDRRGFGQ S SQP T - TGYDYDT FAADLNTVL 84
DCH
EYLGVQGAVHVGH S TGGGEVAYYVARY - - PND PVAKAVL I SAV P PLMVKT ESN PDG - L PK
139
EST-P
AHLG I QGAVHVGHS TGGGEVVRYMARH - - PADKVAKAVL I AAVP PLMVQT PDN PGG - LPK
139
EST-F
EHLDLKEVTLVGFS MGGGDVARY I ARHG - - SARVAGLVL LGAVT PL FGQKPDYPQG - VP L
137
EST-F1
NQLGLDKPLVLGHS LGGAISLALALDH PEA-VSGLVLVAPLT HPQPRLPL
167
BPO-EST E H L G T QGAVHVGHS TGGGEVVRYMARY - - PNDKVAKGVL I AAVP PLMVQT PGNPGG - LPK 140
CPO-P
EALDLRNAVH I GHS TGGGEVARYVANDGQ PAGRVAKAVLVSAV P PLMLKT E SNP EG - LP I
141

CPO-L
EALDLRGAVH I GHS TGGGEVARYVARAE - - PGRVAKAVLVS AVP PVMVKSDTNP DG - L P L
139
HPO-R
AHLGLRDVVHVGH S TGGGEVARYVARHG - - AGRVAKAVL I GAVP PLMVQTE SN P EG - L PV
137
BPO-A1
TDLDLRDVTLVAHS MGGGELARYVGRHG - - TGR LR S AVL L SA I P PVMI KSDKN PDG - V PD
137
BPO-A2
ET LDLQDAVLVGF S MGTGEVARYVS SYG - - TAR I AKVA F LAS LE P F LLKTDDNPDGAAP Q
142
DCH
EVFDDLQNQL FKNR SQ FYHDVP AGP FYG FNR P - GAKVS EPVVLNWWR 191
EST-P
SVFDG FQAQVAS NRAQFYRDV PAGP FYGYNR P - GVDA S EGIIGNWWR 191
EST-F
DVFARFKTELLKDRAQF I SDFNA- PFYGINK- -GQVVS QGVQTQT LQ 187
EST-F1
VFWS LAVRPAWLRRFVANTLTVPMGL-LTRRSVVKGVF APDAA P EDF ATRGGGL 220
BPO-EST S V F D D F QVQVATNRAQ FYRDVP SG P FYGYNR P - GAKS S

E G V I G N W W R 192
CPO-P
EVFDG FRKALADNRAQF FLDVP TGP FYG FNRA - GATVH QGVIRNWWR 193
CPO-L
EVFDE FRAALAANRAQ FY I DVP SGP FYG FNRE - GATV S QGLIDHWWL 191
HPO-R
EVFDGFREAVVTNRSQFYLDLASGPFYGFNRP -GADI S QGVIQNWWR 189
BPO-A1

EVFDALKNGVL TER SQFWKDTAEG F F - SANR P - GNKVT QGNKDAFWY 188
BPO-A2
E F FDG I VAAVKADRYA FYTGF FND - FYNLDENLGTR I S EEAVRNSWN 194
DCH
QGMMGGAKAHYDG I VAF SQTD F T E AL - - - KK I EV PVL I LHGEDD QVVP F E I S GKKSAE LV
242
EST-P
QGMI GSAKAHYDG I VAF SQTDF TEDL - - - KG I TQP VLVMHGDDD Q I VPYENSGLLSAKLL
242
EST-F
I AL LA S LKATVDCVTAFAE TDF R PDM- - - AK I DVP T LV I HGDGD Q I VP FE TTGKVAAE L I
238
EST-F1
LGMRPDN FYAA S SE I ALVNDCL PGMVKRY PQLAL P I GL I YGAQD KVLDF RRHGQALADKV
280
BPO-EST Q G M I G S AKAHYDGVVAF SQTDF TEDL - - - KK I QQPVLVMHGDDD QI VPYENSGPLSAKLL 243
CPO-P
QGMEGS AKAHYDG I KAF S E TDQT EDL - - - KS I TV P T LVL HGEDD Q I VP I ADAALKS I KLL
244
CPO-L
QGMMGAANAHYEC I AAF S E TDF TDDL - - - KR I DV PVLVAHGTDD QVVPYADAA PKSAEL L
242
HPO-R
QGMTGS AQAHYEG I KAF SE TDF TDDL - - - RA I DVP T L I MHGDDD QI VP I ANSAETAVTLV
240
BPO-A1
MAMAQT I EGGVRCVDAFGYTDF TEDL - - - KKFD I PT LVVHGDDD QVVP I DATGRKSAQ I I
239
BPO-A2
TAASGG F FAAAAAP T TW- YTD FRAD I PRIDVPALILHGTGD RT LP I ENTARV FHKAL

244
DCH
KNGKL I SYPG F PH G- -MPTTEAETINKDLLAF I RS
275
EST-P
PNGT LKTYQGYP H G - - MP TTHADV I NADL LAF I R S
275
EST-F
KGAE LKVYKDAPH G - - FAVTHAQQLNEDL LA F LKR
271
EST-F1
PGLKLQVVEGRGH M- - L P I TATARVVEAVLHVAKRVR PVE TATVLHP P FALANK
332
BPO-EST P N G T L K T Y K G F P H G

MP T THADV I NADLVA F I R S
276
CPO-P
QNGT LKTY PGYSH G - - MLTVNADVLNADL LAFVQA
277
CPO-L
ANATLKSYEGL PH G- -MLS THPEVLNPDLLAFVKS
275
HPO-R
KNARLKVYPGL SH G - - MCT VNADTVNADL L S F I E S
273
BPO-A1
PNAE LKVYEGS SH G I AMVPGDKEKFNRDL L E FLNK
274
BPO-A2

PSAEYVEVEGAPH G- - LLWTHAEEVNTALLAFLAK
277
Fig. 5. Alignment of DCH of A. calc oaceticu s F46 with several serine-hydrolases and perhydrolases. The deduced amino acid sequences of DCH, the
esterases of P. putida MR-2068 (EST-P) [25], P. fluorescence SIK WI (EST-F) [12,26], and P. fluorescence DSM 50106 (EST-F1) [27], and the
perhydrolase of P. putida (BPO-EST) [28], P. pyrrocinia (CPO-P) [29], S. lividans (CPO-L) [7], R. erythropolis (HPO-R) [8], and S. aureofaciens
(BPO-A1, BPO-A2) [30,31] are aligned. Identical amino acid residues are enclosed in boxes. Ser, Asn and His residues which are thought to be
involved in the catalytic triad are denoted by black boxes.
Ó FEBS 2003 Role of 3,4-dihydrocoumarin hydrolase of Acinetobacter (Eur. J. Biochem. 270) 491
Sensitivity to peroxides
The tolerance of each strain to peroxides, i.e. peracetic acid
and hydrogen peroxide, was measured by means of disk
inhibition assays (Table 2). The dchD strain was more
sensitive than the wild-type strain to peracetic acid. On the
other hand, E. coli expressing DCH showed greater toler-
ance to peracetic acid than E. coli bearing pKK223-3, but
the difference in the diameters of growth inhibition zones of
transformed E. coli strains was not appreciable in contrast
with those of Acinetobacter strains. For this reason, it was
assumed that E. coli JM109 itself possessed a certain
tolerance to peroxoacids. The sensitivities to hydrogen
peroxide and acetic acid were not affected in either
Acinetobacter strain (dchD or wild-type) or E. coli strains
(pDCH21 and pKK223-3 transformants).
Discussion
In the previous study, we found that the amino acid
sequences of the N-terminal and internal peptide of DCH
exhibited significant similarity to those of several hydrolases
and perhydrolase, and that DCH was a bifunctional enzyme
capable of both hydrolysis of ester bonds and halogenation
[1]. The results of nucleotide sequencing analysis of dch were

also in good agreement with this observation. The active site
amino acid residues of serine-hydrolases, the so-called
catalytic triad, consisting of the consensus motif Gly-X-
Ser-X-Gly and Asp and His residues, were highly conserved
in the deduced amino acid sequences of dch and homolog-
ous proteins, and these enzymes were suggested to belong to
the serine-ÔhydrolaseÕ family. On the other hand, the
recombinant E. coli expressing dch showed not only
lactone-hydrolysing activity but also monochlorodimedon-
brominating activity. This fact indicates that both the
hydrolysis of ester compounds and the halogenation of
organic compounds must be catalysed by the same enzyme.
In fact, several bifunctional enzymes, other than DCH, that
catalyse both hydrolysis and hologenating reactions have
already been reported [26,27]. However, the physiological
roles of these enzymes were not known. To elucidate the
functions of these bifunctional enzymes, we directed our
1234
97.4
66.3
42.4
30.0
20.1
kDa
Fig. 6. SDS/PAGE of the purified DCH and cell-free extracts of
recombinant E. co li strains. Lane 1, purified DCH, 10 lg; lanes 2 and 3,
cell-free extracts of E. coli JM109 bearing pDCH21 and pKK223-3,
respectively; lane 4, molecular mass standards (Daiichi Chemicals,
Tokyo, Japan): phosphorylase b (97.4 kDa), BSA (66.3 kDa), aldo-
lase (42.4 kDa), carbonic anhydrase (30.0 kDa), and trypsin inhibitor

(20.1 kDa). The gel was stained for protein with Coomassie brilliant
blue R-250 and destained in ethanol/acetic acid/water (3 : 1 : 6, v/v/v).
Fig. 7. Northern analysis of total RNA from early log phase A. calco-
aceticus F46 cells. Thrity lg total RNA was loaded on each lane. The
internal gene fragment of either dch (A) or cat (B) was used as a probe.
The approximate sizes of the transcripts are indicated.
492 K. Honda et al. (Eur. J. Biochem. 270) Ó FEBS 2003
attention to the ability of dch to catalyse the peroxidation of
organic acids, which could be an intermediary reaction of
halogenation, and it is assumed that the role of DCH in vivo
is the degradation of peroxoacids.
Peroxoacids are powerful antimicrobial agents like
hydrogen peroxide, and are used for sterilization in several
industrial settings. Anderson and Miller reported that cells
of a plant-colonizing bacterium, P. putida,weremore
sensitive to killing by peracetic acid when they lacked a
major catalase activity [36]. However, it was assumed that
the catalase catalysed the degradation of hydrogen per-
oxide, which was part of the peracetic acid-equilibrium
mixture and did not act directly on peracetic acid. On the
other hand, Picard et al. revealed that a perhydrolase,
CPO-T, isolated from S. aureofaciens, could degrade per-
acetic acid [16] and here we demonstrate that purified DCH
catalysed the decomposition of peracetic acid, in a time- and
dose-dependent manner. Both of these results indicate that
DCH and CPO-T act on peracetic acid itself. Although the
products of the enzyme reaction were not determined in this
study, DCH was thought to convert peracetic acid to acetic
acid and hydrogen peroxide because of the existing evidence
that DCH and other perhydrolases are able to catalyse the

reverse reaction, i.e. the formation of a peracetic acid from
acetic acid and hydrogen peroxide [1,12,16,28] and because
DCH could catalyse the halogenation reaction in the
presence of not only acetic acid but also other organic
acids, such as formic acid, propionic acid, and n-butyric acid
[1]: peroxoacids corresponding to these organic acids might
also serve as substrates of the enzyme.
DCH was originally isolated as a lactonohydrolase,
which was specific for aromatic lactones, such as
3,4-dihydrocoumarin, 2-coumaranone and homogentisic
acid lactone, and considered to participate in the degrada-
tive metabolic pathway of polycyclic aromatic compounds
[1]. However, judging from the K
m
and V
max
values for
peracetic acid and other substrates, peracetic acid is likely to
be a natural substrate for DCH in vivo.AdchD mutant
derived from A. calcoaceticus F46 was more sensitive to
growth inhibition by peracetic acid than the parent strain.
On the other hand, E. coli expressing dch showed increased
resistance to peracetic acid. These results also suggested that
DCH detoxifies peroxoacids and plays a role in the
oxidative stress defence system in vivo. Superoxide dismu-
tase, catalase, peroxidase, etc. are already known to be
antioxidant enzymes, and have been well investigated.
However, enzymes responsible for defence against peroxo-
acids have not yet been found. DCH detoxifies peracetic
acid in a unique manner, i.e. ÔhydrolyticÕ degradation, and

might be a new example of such antioxidant enzymes.
In addition to these observations, a putative catalase
gene was found immediately downstream of dch. Although
the expression of these genes was constitutive and not
induced on the addition of either peracetic acid or
hydrogen peroxide to the culture medium (data not
shown), Northern analysis revealed that they were tran-
scribed as both monocistronic mRNAs and parts of the
same polycistronic mRNA. This indicates that they are
regulated by the same promoter, and DCH and the
catalase might play a cooperative roles in the oxidative
defence system, i.e. at first, peroxoacids are hydrolysed to
the corresponding organic acids and hydrogen peroxide by
DCH, and then the resulting hydrogen peroxide is
degraded by the catalase. In prokaryotes, however, genes
abutting each other tend to be transcribed as a polycis-
tronic mRNA. To confirm this novel enzymatic antioxi-
dative stress system, further investigations on DCH and
other perhydrolases are required. Recently, with the great
advances in genetic analysis, a lot of perhydrolase-like
genes have been found in various bacteria. The novel
oxidative stress defence system presented in this study
might be more widespread in nature than we thought.
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific
Research nos. 01729 (to K. H.) and 14360054 (to M. K.) from the
Japan Society for the Promotion of Science. We also thank T. Ishige,
A.Tani,Y.SakaiandN.Katofortheirhelpinthegenedisruptionof
Acinetobacter.
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