Journal of Environmental Biotechnology
Vol. 20, No. 1, 79–82, 2020

Original paper (regular paper)

Dioxin Reductive Etherase DreE Reductively Cleaves Two Diaryl EtherBonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a Sequential Fashion
Yuzo Suzuki1, Masaya Nakamura1*, Yuichiro Otsuka1, Nao Suzuki2, Keisuke Ohyama2,Takeshi Kawakami3, Kanna Sato-Izawa4, Shojiro Hishiyama1, Kouya Inoue5,Toshiji Kameyama5, Atsushi Takahashi3 and Yoshihiro Katayama6
1
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305–8687, JapanGraduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture & Technology,Koganei, Tokyo 184–8588, Japan
3
Takasago Thermal Engineering Co. Ltd., Shinjyuku, Tokyo 160–0022, Japan
4
Department of Bioscience, Faculty of Life Sciences, Tokyo University of Agriculture, Setagaya, Tokyo 156–8502, Japan
5
Kantteku Co. Ltd., Bunkyo, Tokyo 112–0004, Japan
6
College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–0880, Japan
2* TEL: +81–29–829–8282 FAX: +81–29–873–3797* E-mail address: nmasaya@ffpri.affrc.go.jp
We have previously reported cloning and characterizing of the gene encoding the dioxin reductive etherase DreE for thereductive cleavage of diaryl ether bond of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a dioxin with the highest toxicity, inGeobacillus thermodenitrificans UZO 3. DreE expressed in Escherichia coli could reductively cleave diaryl ether bond of
2,3,7,8-TCDD to produce 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the reaction product. In this study, we
have investigated whether TCDE is first produced as a reaction intermediate which is then converted to 3,4-dichlorophenol(DCP) from 2,3,7,8-TCDD degradation mediated by DreE.
Key words: Bioremediation, Dioxin, 2,3,7,8-TCDD, TCDE, DreE
Abbreviations: 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDE, 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether;
DCP, 3,4-dichlorophenol; dreE, dioxin reductive etherase; PCDDs, polychlorinated dibenzo-p-dioxins;
PCDFs, dibenzofurans; CoA, coenzyme A; CoASSCOA, coenzyme A disulfide; BSTFA, N,O-Bis(trimethylsilyl)trifluoroacetamide; Km, kanamycin.
(Received: 21 August, 2019/Accepted: 16 October, 2019)

Dioxins consisting polychlorinated dibenzo-p-dioxins(PCDDs) and dibenzofurans (PCDFs) have been formed
not only naturally but also unintentionally as a by–products
during the bleaching of pulp and the manufacture of pesticides 5). PCDDs and PCDFs contamination has led to serious
social problem because of their carcinogenic and genotoxic
properties 11). 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8TCDD) is the most toxic among the dioxin isomers 4,6,12).
Microbial degradation of model compounds of PCDDs andPCDFs has been studied using many microorganisms 3,18).
However, despite numerous studies on the microbial degradation of PCDDs and PCDFs, no report has demonstrated thedegradation of 2,3,7,8-TCDD at the molecular level.
We have previously reported cloning the gene encoding
the dioxin reductive etherase DreE from the genome libraryof G. thermodenitrificans UZO 3 and showed that DreE
expressed in E. coli could reductively cleave diaryl etherbond of 2,3,7,8-TCDD to produce TCDE as the reactionproduct 16). Furthermore, DreE was found to have aminoacid homology to RibT, a protein with yet unknown function that is encoded as part of the riboflavin biosynthesis
operon of Bacillus subtilis 13,19). On the other hand, nosignificant amino acid homology was found between DreE
and a known reduction cleavage enzyme, β-etherase (LigE,LigF) of Sphingobium sp. strain SYK-6, that reductively
cleaves the β-aryl ether bond that is the main intramolecularbond of lignin using GSH as electron donor 7–10). These results
suggest that the 2,3,7,8-TCDD degradation reaction is anovel reducing reaction different from known variations that
require electron donors, such as GSH. We also showed thatthe deduced amino acid sequence of DreE possesses a coenzyme A (CoA) binding site and presumed that it is involved in
2,3,7,8-TCDD degradation by DreE. Previously, delCardayre
et al reported that CoA, a coenzyme in various acyl grouptransfer reactions, also functions as a GSH analog and maintains a reductive environment in gram-positive bacteria 1,2,14).
Many gram-positive bacteria do not produce GSH, insteadthese organisms produce CoA at high levels by CoA disul-Suzuki, et al.

80

Cl** OClClOClDreEClHOClCl**TCDEClO

2,3,7,8-TCDDDreEClOH

Cl+ClHOCl

2 × DCP

2CoACoASSCoA

2CoACoASSCoA

2NADP+

2NADPH

2NADP+2NADPH

CoA disulfide reductase

CoA disulfide reductase

Fig. 1. Proposed degradation pathways for 2,3,7,8-TCDD by DreE. Enzyme: DreE, dioxin reductive etherase. Abbreviations: 2,3,7,8-TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDE, 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether; DCP, 3,4-dichlorophenol; CoA, coenzyme A;
CoASSCOA, coenzyme A disulfide. Asterisks indicate the positions where hydrogen is added.

AB
3Abundance (106)Abundance (107)

1.5
10.5

ClTMSO

ClClO

Cl

TCDE(%)
10021950 73

234

20100200

1DCP
040050

C
1.5

10.5

ClTMSO

ClClO

Cl

TCDE

40(%)
10050

100.5

23473
100200E400

ClTMSO

ClClO

Cl

Cl

TMSO

ClClO

Cl

Cl

40

TCDE50F
1.5

10.5030300

ClTMSO

050

Abundance (107)Abundance (107)1.5403050219DCP0400Cl30D
Abundance (107)Abundance (107)1.530300

ClTMSO

30ClTMSO

ClClOClTCDE

40Retention time (min)50

10.503040Retention time (min)TCDE

50Fig. 2. GC-MS analyses of the reaction mixtures for TCDE degradation mediated by DreE. The selected ion monitoring (SIM) chromatograms of the substrate TCDE at m/z=396 (C), m/z=234 (D) and the detected product DCP at m/z=234 (D), and its corresponding MS
spectrum were compared to authentic TCDE and DCP (A, B). The SIM chromatograms at m/z=396 (E) and at m/z=234 (F) represent
the control chromatograms obtained when DreE was lacking from the enzyme assay.

DreE cleaves two diaryl ether bonds of 2,3,7,8-TCDD

fide reductases. Thus, it was suggested that CoA functions
as a GSH analog in G. thermodenitrificans UZO 3 and is
involved in 2,3,7,8-TCDD degradation by DreE. We therefore have proposed a molecular mechanism model for thereductive cleavage of diaryl ether bonds of 2,3,7,8-TCDDin G. thermodenitrificans UZO 3 (Fig. 1). DreE reductively
cleaves diaryl ether bonds of 2,3,7,8-TCDD using two hydrogens from two molecules of CoA, which acts as an electron
donor. Furthermore, CoASSCoA (CoA disulfide) produced
from the reaction may be NADPH-dependently converted to
two molecules of CoA by CoA disulfide reductase, which ispredicted to exist in G. thermodenitrificans UZO 3.
However, contrary to our previous data with cell-free
extract prepared from a wild strain of G. thermodenitrificans UZO 17), DCP was not generated as a final productfrom 2,3,7,8-TCDD degradation mediated by DreE. In thisstudy, we have investigated whether TCDE is first produced
as a reaction intermediate which is then converted to DCP
from 2,3,7,8-TCDD degradation mediated by DreE (Fig. 1).
The TCDE was prepared following previous protocols 16,17). DCP, N,O-Bis(trimethyl silyl)trifluoroacetamide(BSTFA), kanamycin (Km) and diethyl ether were purchasedfrom Wako (Osaka). E. coli BL21 (DE3) was used as thehost strain 15). The expression plasmid pETE in which thedreE gene is inserted downstream of the T7 promoter ofpET28b(+) (Novagen) was isolated as in a previous study 16).
Luria-Bertani medium was utilized for the cultivation ofE. coli strains. For the culture of cells carrying antibiotic
resistance markers, the media for E. coli transformants wassupplemented with 25 mg of Km/liter. DreE preparation,
the enzymatic reaction assays and GC-MS analysis wereperformed as described previously 16,17). Briefly, 1 mL reaction
mixture containing DreE (200 μg of protein/mL) and 15 μM
TCDE dissolved in toluene and acetone (final concentration
0.5% and 2%, respectively) was prepared. The enzymatic
reaction was performed at 65°C for 2 h inside a controlled
incubator. Diethyl ether extracts from the TCDE reaction
mixture were dried. The dried enzyme reactant was derivatized by BSTFA prior to GC-MS analysis.
We analyzed the TCDE-degrading activity of DreE toinvestigate whether it was a reaction intermediate during
2,3,7,8-TCDD degradation to DCP. GC-MS analysis of thediethyl ether extract of the complete reaction mixture inaddition to the substrate TCDE showed peaks for DCPat retention times 27.45 min (Fig. 2D). The peaks of thesubstrate and products corresponded well with those of theirrespective authentic compounds (Fig. 2A, B). Diethyl ether
extract of a reaction mixture that did not contain the DreE(control) was detected of TCDE, but showed no peak for
DCP (Fig. 2E, F). These results show that DreE reductive
cleaves of the two diaryl ether bonds of 2,3,7,8-TCDD in asequential fashion. That is, TCDE is first produced by DreE
as a reaction intermediate which is then converted to DCP(Fig. 1).

81

Acknowledgements
This work was supported by MEXT KAKENHI Grant
Number JP21248037, JSPS KAKENHI Grant Number
JP12J00579 and a fund M-02 from Ministry of theEnvironment of Japan.
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