Cl'.iorinavec! ;!a Lori^n-p-:;: o;:ir.s
Texas Univ. at .Vj.-r.tin
pypcrad for
Environracntal P.c?sc:
Gulf Crecz:e, FL
Bar 81
i
aCTgza^^
v/vsws^^
C.
t.. .
— 81-016
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants required that regulations restricting the introduction
of these compounds into the environment be formulated on a sound scientific
basis. Accurate information describing dose-response relationships for organisms
and ecosystems under varying conditions is required. Tlie Environmental Research
Laboratory, Gulf Breeze contirbutes to this infoniistion through research programs
aisned at determining:
. the effects of toxic organic pollutants on individual species and
communities of organisms;
. the effects of toxic organics on ecosystem processes and components;
. the significance of chemical carcinogens in the estuarine and marina
environment.
This report sought to determine the initial oxidative pathways for
dibenzodioxin and diuenzofuran compounds by bacteria in aouatic systems.
Bacterial cultures were isolated and oxidized dibenzo-p-dioxin and nano-
chlorinated dibenzo-p-dioxins; however, di-chlorinated dibenzo-p-dioxins,
were not metabolized by either strain. The metabolite, 1,2-dihydrodibenzo-p-diosin,
was a potent inhibitor of further oxidation. These results vri'H sld ir. the
understanding of bacterial metabolic mechanisms of aromatic eompsfunds and
eventually may help to predict their fate in aquatic onvironmsnts.
Henry FO'tr.os
Director
Envirorsnental Research Laboratory
Gulf Breeze, Florida
iii
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ABSTRACT
Peeudomanas sp. N.C.I.B. 9816, strain 11, when grown on salicylate in the
presence of dibenzo-p-tiicxin, accumulated do-} ,2-dinytiroxy-l ,2-diiiycrodiben-
zo-p-dioxin and 2-hydroxydibc-nzo-p-dioxin in the culture medium. E?.ch r.etabo-
lite v.'as isolated in crystalline fonn and identified by a variety of conven-
tional chemical techniques. Crude coll extracts prepared frem the prrental
strain grown with naphthalene oxidised cie-1 ,2-iiinydroxy-l ^-tl-ihydrcoiLr.-nzo-
p-dioxin under both aerobic and anaerobic conditions to 1,2-dihydrorycib^riZo-
p-dioxin. Further degradation of this metabolite was not det?cteci.
Whole cells of the parent strain of Beijcriwkia, grcv.-n v:ith succinate
and biphenyl, oxidized dibenzo-p-dioxin and several chlorinated dicxins. The
rete of oxidation of the chlorinated dib-nzo-p-dioxins dscre-sed v;ith r.n in-
creasing degree of chlorine substitution. A mutant strain (E3/"S) of h,-iji,r-
inakia oxidized dibenzo-p-dioxin to c-ic-1,2-dihydroxy-l ,2-dihydrcc^benzo-p-
dioxin. The mutant organism also oxidized teo moriocn'ioriiiated dit"znzo-p-
dioxins to cts-dihydrodiols. No metabolites were detected frc.-i t\.'0 dichlorin-
ated dibenzo-p-dioxins. Growth of the parent strain of Bcijeri~j-a- present
in the culture rrsdium. Resting cell suspensions of the parent. O^^HTST], pre-
viously grown with succinate and biphenyl, oxidized dibenzo-p-dio;:in to a
compound identified as 1.2-dihydroxydibenzo-p-dioxin. Further depraoatirn of
this metabolite was not detected, as the compound was found to be <; pc^jnt
mixed-type inhibitor of tv:o ring-fission oxygenases present in this organism.
This report wss submitted in fulfillment of Grant No. R-8CH525 by The
University of Texas under partial sponsorship of the U.S. Environmental Pro-
tection Agency. This report covers the period from December 1. 1976 to
November 30, 1979 and was completed as of December 7, 1979.
IV
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EPA 600/4-81-016
torch 1981
BACTERIAL DEGRADATION OF DIBENZO-£-DlOXIN
AND CHLORINATED DIBENZO-£-DIOXINS
by
G.M. Klecka and D.T. Gibson
Department of Microbiology
The University of Texas at Austin
Austin, Texas 78712
Grant No. R-804525
Project Officer
AT W. Bourquin
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32551
U.S. Environmental Protection Agency
Office of Research and Development
Environmental Research Laboratory
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviev:3d by the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Afency, and approved vor publication.
Approval does not signify that the contents necessarily reflect the viev.'s and
policies of the U.S. Environmental Protection A
-------
1. ficKJRT NO.
rcn::r;'CAL RE-KWT ri
(F.tsxnodiiuinici>&a ox ilie/rezrt* K!. AUTHOR C>)
G.M. Klecka and D.T. Gibson
COD2
.'l2AYlGiU KA«£ AWO ADDRESS
1<3. ,'itOGHAtj! ELUli;i.
11. C
Al-* V k
Grant Mo. R-504525
12. SPONSORING AaE
U.S. Environmental Protection Agency
Office of Researach and Davelopirsant
Environmental Research Laboratory
Gulf Breeze, Florida 32551 _
13. TYPE 0? REPORT t-SlQ PCBtOO COVERED
Final - Dsc. 19/5. - iiov. 197?
14. s.«>owsoRir*e Aoewcv CODE
CPA-600/4
IS. SUFPLtiMENTAKY HOT63
IS. ABSTRACT
Pseua'Q!?ion5s sp. N.C.I.B. 9816, strain 11, when grown on salicylate in the pres-
ence ot cnbenzo-n-dioxin, accumulated cj^-l^-dihydroxy-l^-dihydrodibenzo-^dioxin
and 2-hydroxydibenzo-D-dioxin In the culture insdiisn. f,rude cell ext. acts prepared
from the parental strain grown with naphthalene oxidized ci s-1 ,^-dl hydroxy-1 , 2-
dihydrodibcnzo-p_-dioxin to l,2-dihydroxydibenzo-2-dicxon. Further degradation of
the metabolite was not detected. Whole cell-- of the parent strain of Bgi.-jgrinckia,
grown v-iith succinate and biphenyl, oxidized dibenzo-p-dioxin and several
dioxins. A mutant strain (B8/36) of Eai.isrinicka oxfdized dibenzo-^-dioxin to cis-
l,2-dihydroxy-l,2-dihydrodibfinzo-p-dioxm. 'me mutant organism also oxidized two
nonochlorinated dibenzo-p-dioxins to cj[s_-diiwdrodio!$. No mstabolites v=:ere detected
frcsi tv,'o dichlorinated dibenzo-p^-dicxins. Growth of the parent strain of Bei jerirr.cki
on succinate was inhibited after four hours when 0.05% dibenzo-£-dioxin was present
in the culture msdiimi. Resting cell suspensions of the parent organism oxidized
dibenzo-g-dioxin to a compound identified as 1,2-dihydroxyciibenzo-p-dioxin. Further
degradation of this metabolite was not detected, as the compound was found to be a
potent mixed-type inhibitor of two ring-fission oxygenases present in this organism.
VI.
i.
KEY WORD3 AMD DOC'JWEHT ANALYSIS
EMDiO TERMS JC. COSATI F
Chlorinated dibenzo-p-dioxins
Hicrobial degradation
ci s-di hydrodi ols
inhibition of bacterial growth
10. DiaTH'JiUTIOH I.V/
Release to public
13. SECURITY CLAiS (Vni* ntporlj
'l. HO. Of-
i-D. EECUFitTY CLA-i
. Unclassified
ZX. CRIC6
^jr
PA Fom 2220-f (»«». 4-77) PREVIOUJ CUITIO
i^oyri c<:
NATIOhiM. T'-CHMCAl
INFORMATION SERVICE
uj titMuitaiii of cofieiFci
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CONTENTS
Forev;ord ill
Abstract iv
Figures vi
Tables ..... viil
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Materials end Methods 5
5. Results and Discussion 12
References 52
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FIGURES
Number
1 Thin-layer chromatogram of the products formed from the
oxidation of dibenzo-p_-dioxin by Pseudomonas sp. N.C.I.B.
9816 strain 11 16
2 Formation of compound A by Pseudomonas sp. N.C.I.B, 9816
strain 11 17
3 Infrared spectrum of compound B (Nujol) 18
4 Ultraviolet spectrum of compound B (MeOH) 19
5 Proton magnetic resonance spectrum of compound B (Acetone-
d5) 20
6 Infrared spectrum of compound A (Nujol) 22
7 Ultraviolet spectrum of compound A (MeOH) 23
8 Proton magnetic resonance spectrum of compound A (Pyridine-
d5) 24
9 Infrared spectrum of compound C (Nujol) 27
10 Ultraviolet spectrum of compound C (KeOH) 28
11 Proton magnetic resonance spectrum of compound C (CDClg) ... 29
12 Infrared spectrum of compound D (Nujol) 32
13 Ultraviolet spectrum of compound D (MeOH) 33
14 Proton magnetic resonance spectrum of compound D (Acetone-
d6) 34
15 Polarographic assay of 1,2-dihydroxynaphthalene oxygenase
in cell-free extracts of Pseudomonas sp. N.C.I.B. 9816 . . . 36
16 Growth and acci-mulation of compound IA by cultures of
Beijerinckia B8/36 39
vi
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IJumber Page
17 Growth and accumulation of ccirioounds IIA end IIIA by
cultures of Beijerinckia E8/35 41
18 Acid-catalyzed dehydration of compounds IIA and IIIA 43
19 Effect of dibenzo-£-dioxin on the growth of Bgijerinckia
sp., wild type 45
20 Polarographic assay of ring-fission oxygsnases in cell
extracts of Deijerinckia sp 45
21 Separation of the 2,3-dihydro;rybip!-,onvl ^ Cctechol
oxygenar.cs by ion-exchange (CEAE) cnrcmatography 48
22 Effect of 1,2-dihydroxydibenzo-D-dioxin on the ring-
fission oxygenases from Beijerinckia sp 51
v.i
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TABLES
Number
1 Sugary of the preliminary screening of organisms for the
ability to co-oxidize dibenzo-£-dioxin 13
2 Oxidation of substrate's by washed, whole cells of Pseudo-
monas sp. N.C.I.B. 9816 ." . . . 14
3 Analysis of the proton magnetic resonance spectrum of
compound B 21
4 Analysis of the proton magnetic resonance spectrum of
compound A 25
5 Analysis of the proton magnetic resonance spectrum of
compound C 30
6 Analysis of the proton magnetic resonance spectrum of
compound D 35
7 Oxidation of substrates by washed, whole cells of
Beijerinckia sp 37
8 Physical properties of the metabolites formed from mono-
chlorinated dibenzo-g_-dioxins by Beiierinckia B8/36 42
9 Polarographic analysis of the activities of the partially
purified 2,3-dihydroxybiphenyl a;id catechol oxygenases ... 50
vi i i
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SECTION 1
INTRODUCTION
Heterocyclic aromatic compounds containing 2 atcrns of oxygen, such as
dibenzo-p_-dioxin have been identified in tha neutral fractions of bituminous
coal (16). Interest in chlorinated dibenzo-p_-dioxins arose with the identi-
fication by x-ray diffraction of 1,2,3,7,3,9-hexachlorodibenzo-p_-dioxin as
the "chick edama fector" which caused the deaths cf millions of broiler
chickens in 1957 (4). It is now known that conditions utilized in the syn-
thesis of certain chlorinated fungicides and herbicides, such as pentachloro-
phenol, 2,4,5-trichlorophenol and 2,4,5-trichlorophenoxyacetic acid can re-
sult in the formation of chlorinated dibenzo-£-dioxins. The most potent iso-
mer, 2,3,7,8-tetrachlorodibenzo-p_-dioxin has been considered to be one of the
most toxic low molecular weight compounds known to man, with a me?.n lethal
dose in guinea pigs of 1.0 ug/kg of body weight (27). The high toxicity of
some of these contaminants, when considered in light of the extensive world-
wide use of chlorophenols 'In both industry and agricul lure, is of environ-
mental concern.
The potential for the degradation of chlorinated dibenzo-p_-dicxins in
the environment has been investigated. Reductive dechlorination has been re-
ported by several authors (7,8). The reaction required ultraviolet light and
the presence of a suitable organic hydrogen donor. Kearney et_ al_. O8) have
monitored the metabolism and persistence of ^C-labelled 2,7-dichlorodibenzo-
p_-dioxin and 2,3,7,8-tetrachlorodibenzo-p_-dioxin in experimental soil samples.
-------
2,7-Dichlo>*odibfw.o-p_-dioxin was oxidized to -COg and to cne or mere polar
metabolites detected in ethanol extracts of the soil samples. Attempts to
identify the metabolites were unsuccessful. 2,3,7,8-Tetrachlorodibenzc-£-
dioxin was oxidized to a lesser extent, as over 50% of the original samples
were recovered after one year. No metabolites were <1etec .l.ed in soils treated
with 2,3,7,8-tetrachlorodibenzo-£-dioxin. Matsumura & Benezet (21) screened
over 100 inicrcbial strains previously shown to be capable of degrading per-
sistent pesticides for the ability to degrade ^C-labelled 2,3,/,8-tetra-
ci-ilorodibenzo-£-dioxin. Only 5 strains were found to be capable of degrauing
this compound to unidentified polar metabolites.
This report describes the oxidation of dibenzo-p^dioxin and severs!
chlorinated dibenz^-p_-dioxins by a Pseudcmonas species and a 8ei jen'nckie spr-
cies. Evidence is presented for the identity of metabolites prior to the en-
zymic fission of the aromatic nucleus. Further eviuencn is prerented that
describes the inhibition by 1,2-dihydroxydibenzo-p_-dioxin of enzymes respon-
sible for the oxidative fission of arotr.^tic molecules.
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SECTION 2
ccficLusions
The results cf laboratory studies on the biedegr^.'ation of dibc.'n;'0-^-
dioxin and its chlorinated derivatives suggest that rcicrcoroanisms capable of
oxidizing these compounds are relatively rare in the environment. The unsub-
stituted parent compound, dibenzo-p_-dioxin, was shown to inhibit the growth of
a laboratory strain of Bei.ierinckia and this physiological effect rray be due
to the rretabolism of dibenzo-£-dioxin to 1,2-dihydroxydibenzo-p_-u'ioxiri. The
latter compounJ was shown to be a potent inhibitor of ring-fission dioyyr,enases
in this organism.
Dibenzo-p_-dioxin and two monochlorinated derivatives were oxidized to cis-
dihydrodiols by a Beijerinckia species. Pseudomonas sp. U.C.I.B. 981G also
oxidized dibenzo-p_-dioy,'in to a ci^-dihydrodiol. These observations indicate
that microorganisms capable of oxidizing this class of compounds use a dioxy-
genase enzyme system to incorporate rrolecular oxygen into the aromatic nucleus.
This type of metabolism is similar to that observed in the biodeqradation of
aromatic hydrocarbons and related compounds. Increasing chlorine substitution
on the aromatic rings of dibenzo~£-dioxin decreases the rate of biological
oxidation.
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SECTION 3
RECCOEDNATIONS
It is recommended that further studies on the microbial oxidation of
dibenzo-£-dioxin and its chlorinated derivatives be conducted. Preliminary
observations suggest that dibenzo-^-dioxin itself inhibits the growth of bac-
teria and the mechanism of this inhibition should be elucidated. A broader
range of microorganisms including bacteria, fungi, yeasts and alga, should be
examined for their ability to oxidize this class of compounds in order to de-
termine the potential fate and effects of dioxins in the environment.
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SECTION 4
MATERIALS AT1D I'ETKODS
Organisms, and Grovfth Conditions
Four strains of bacteria vvere examined for the ability to cxidi/e diben-
zo-£-dioxin and chlorinated dibenzo-p_-dioxins. Pscbdrrtrir? sp. N.C.I.B. was
kindly provided by Professor W.C. Evans, University of North '..'ales. Great
Britain. The organism was isolated from farm soil by elective culture, and
has the ability to grow on naphthalene as the sole source of carbon (9). A
spontaneous mutant, Pseudomonas sp. N.C.I.B. 9816 strain 11, was isolated from
the parent organism, that co-oxidized naphthalene to cis-1,2-dlhydroxy-l ,2-
dihydronaphthalene when grown on succinate or salicylate as the carbon source
(D.T. Gibson, unpublished results).
The isolation and characterization of Bei jc-rinckia sp. has been reported
(13). The organism was isolated from a polluted stream by virtue of its abil-
ity to groH with biphenyl as a sole source of carbon. Treatment of the parent
organism with fi-methyl-N'-nitro-N-nitrosoguanidine led to the isolation of a
mutant strain, Beijerinckia 88/35, that oxidizes several different aromatic
hydrocarbons to ci_s-dihydrodiols (12,13,17,19).
Organisms were maintained on agar slants of a mineral salts medium (29)
containing 0.2% succinate, and were stored at 4 C. Cells were transferred
from a slant to 100 nil of mineral salts broth (pH 7.2) containing 0.21 suc-
cinate in a 500 ml Erlermeyer flask. Cultures were incubated at 27 C on a
rotary shaker at 150 rpm for 12 hours. A 5% (v/v) inoculum of the aoove
-------
culture was used for further experiments.
Biotransformation experiments \vere conducted in 2 liter Erlenmoyer flasks
containing 500 ml of mineral salts medium as above, with the exception that
the phosphate buffering capacity was double strength ;pH 7.2). Carbon sources
were added to the culture medium according to the experimental design. Naph-
thalene, biphenyl, snd dibenzo-2-dioxins were added to cultures in 10.0 ml of
acetone to give 3 final concentration of 0.05% in the culture medium. Acetone
was not a growth substrate nor did it inhibit the growth of the organisms.
Flasks were incubated as described above. Growth of the cultures was moni-
tored turbidometrically at 600 nm. When samples of the cultures were filtered
through glass wool, the aromatic hydrocarbons did not interfere with the mea-
surement of cell density.
Large-scale biotransforaation experiments with Pseudomonas sp. N.C.I.B.
9816 were conducted in a 50 liter carboy containing 10 liters of medium with
0.1* salicylate and 0.05% dibenzo-p_-dioxin. Cultures were incubated on a re-
ciprocating shaker at 27 C.
Cells of Pseudumonas sp. K.C.I.B. 9816 and Beijerinckia sp. used for the
preparation of cell extracts were obtained from large-scale incubations (10
liter) grown with forced aeration in a New Brunswick model H14 Microferm fer-
mentor at 30 C for 12 hours. Air was supplied at a rate of 10 liters/mln and
the culture was stirred at 500 rpm. Pseudomonas sp. N.C.I.B. 9816 was grown
with 0.1» naphthalene. Beijerinckia sp. was grown with 0.2% succinate and
0.052 biphenyl as an inducing substrate. In addition, an anion exchange resin
(BioRad AG 1-X3: 5 g/1) was added to the latter cultures to remove acid prod-
ucts (19). Cultures were initially filtered through glass wool to remove
solid materials, and the cells harvested with an air-driven Sharpies continu-
-------
ous-flow centrifuge. The cells were washed twice with 0.05 M KH2PO/( buffer,
pH 7.2, and used in further studies. Unused cells were stored at -15 C until
required.
Detection of Metabolites
Quantitative measurement of product accumulation in the culture medium
v;as conducted at various time intervals. A 5.0 nil aliquot of the culture was
extracted with 15.0 ml of ethyl acetate. The organic extract was dried over
anhydrous Na2S04 and the solvent removed in vecuo at 30 C. The residue w^s
applied with 0.5 ml of acetone to a glass preparative thin-layer chrcmatogra-
phy plate, precoated with Silica-Gal 60. The solvent system used for chrcrna-
tography was chloroform/acetone (80:20). Compounds were located with the use
of ultraviolet light. Regions of the plate containing suspected metabolites
were removed and extracted with 5.0 ml of methanol. A sample (0.1 ml) of the
methanol extract was diluted to 1.0 ml with the sarre solvent and the ultra-
violet spectrum recorded. The reference cuvette contained methanol.
Alternatively, samples of duplicate culture extracts were examined on
analytical thin-layer chrornatography sheets. Following development of the
chromatograms as above, compounds were located with the use of ultraviolet
light and also by spraying with a 2% solution of 2,6-dichloroquinone-4-
chloroimide in methanol (10; Gibbs reagent). Subsequent exposure of the chro-
matogram to ammonia vapors often enhanced the color intensities of the differ-
ent products.
Isolation of Transformation Products
When product accumulation in a culture had reached a maximum, the culture
was filtered through glass wool to remove unused solid materials. The fil-
trate was extracted with two volumes of ethyl acetate and the organic layer
-------
dried over anhydrous ^SC^. The solvent was removed IP vacuo at 30 C. Prod-
ucts were purified by column chr&Tiatography followed by recrystallization.
Whole Cell Studies
Determination of the rate of whole cell oxidation of various substrates
was conducted polarographically with a Clark-type oxygen electrode at 30 C,
using 0.05 M KK2P04 buffer, pH 7.0, which was saturated with air prior to use.
The reaction mixture, in a total volume of 1.4 ml contained buffer and an ali-
quot of a cell suspension. The reaction was initiated with the addition of
200 nmoles of the substrate in 10 pi of N.N-dimethylformamide. All rates were
corrected for endogenous respiration of the cell suspension. Whole cells were
unable to oxidize N,N-dimethylformamide nor did this solvent inhibit enzymatic
activity.
Preparation of Cell Extracts
Frozen cells were thawed and suspended in 0.05 M KH2pC)4 buffer, pH 7.2,
containing 10% acetone (3 g of cells per 10 ml of buffer). The suspension was
subjected to sonic oscillation at 40 kHz with a Bronwill Biosonik III ultra-
sonic disintegrator for 1.5 min (three 30-sec exposures). Cell debris was re-
moved after centrifugation at 30000 x g for 1 hour at 5 C. The clear super-
natant solution was used as a source of crude cell extract.
Enzyme Analyses
The activity of various enzymes was determined with cell-free extracts.
In all cases, the amount of cell extract used in the analysis was a level
found to give a linear rate of product formation with respect to protein con-
centration.
1,2-Dihydrox.ynap.hthalene Oxyqenase—The activity of the 1,2-dinydroxy-
naphthalene oxygenase was determined polarographically at 30 C using
-------
0.05 M KH2P04 buffer, pH 7.0. The reaction vessel, in a total volume of 1.4 ml
contained buffer and a suitable amount cf cell extract. Reactions were initi-
ated with the addition of 100 nmoles of 1,2-dihydroxynaphthalene.
2,3-Dihydroxybinhenvl Oxyqgnase--Activity of the 2,3-dihydroxybiph2nyl
oxygenase was determined polarographically at 30 C using 0.05 M KI^PC^ buffer,
pH 7.5. The reaction mixture, in e total volume of 1.4 ml contained buffer
and a suitable amount of cell extract. Reactions v/ere initiated v:ith the ad-
dition of 100 nmoles of 2,3-dihydroxybiphenyl in lOul of f!,li-aitr.3thylformsmide.
All rates were corrected for endogenous respiration. No auto-oxidatio;i of
2,3-dihydroxybiphenyl was observed under the assay conditions.
Spectrophotomatric assay conditions v/ere established for the 2,3-dihy-
droxybiphenyl oxygenase. The product of the reaction has been identified as
2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (5) and was found to exhibit an ab-
sorption maximum at 435 nm (e = 15800; pH 7.5) (D.T. Gibson, unpublished re-
sults). The reaction mixture, in a total volume of 1.0 ml contained buffer
and cell extract. Reactions were initiated with the addition of 100 n-noles
of 2,3-dihydroxybiphenyl.
Catechol-2,3-dioxy.ienase--The activity of the catechol-2,3-dioxygenase
(E.G. 1.13.1.2) was determined polarographically at 30 C using 0.05 M KH2PC»4
buffer, pH 7.5. The reaction mixture, in a total volume of 1.4 ml contained
buffer and a suitable amount of cell extract. Reactions were initiated with
the addition of 1.0 wnole of catechol. All rates were corrected for endogen-
ous respiration.
Catechol-2,3-dioxygenase activity was monitored spectrophotometrically
by the procedure described by Nozaki et el. (22) by measuring the formation
of a-hydroxymuconic setnialdehyde at 375 nm. The reaction mixture, in a total
-------
volume of KO ml, contained buffer and cell extract. Reactions were initiated
with the addition of 1.0 yrcole of catechol.
Protein Determinations
Whole cell protein determinations were conducted after digestion of an
aliquot of the cell suspension in 0.1 N NaOH at 100 C for 1 hour. Protein
.concentration was determined by the method of Lowry et al. (20) with crystal-
line bovine serum albumin as the star.uard.
Analytical Methods
Ultraviolet and visible spectra we're determined on a Beckman model 25
recording spectrophotometer. Infrared spectra were recorded on a Perkin-Elmar
model "137 spectrophotometer and were referenced to the absorptions of polysty-
rene. Crystalline samples were mulled in Nujol and placed between NaCl discs.
Optical activity was determined with a Perkin-Elmer model 141 polarimeter.
Low-resolution mass spectra were determined on a DuPont-Consolidated Electro-
dynamics Corp. model 21-491 spectrometer. High-resolution mass spectra were
determined on a DuPont-Consolidated Electrodynamics Corp. model 21-1COOC spec-
trometer and were referenced to assigned perfluoroalkane peak fragments. Pro-
ton magnetic re-- — spectra were recorded on a Varian model HA-100 spectro-
meter. Absorptions »ere assigned 6 values at the mid-poir.t of half-height and
are referenced to tetramethylsilane (Me^Si). Melting points were obtained
with the use of a Biichi melting point apparatus and are uncorrected.
Chemicals and Materials
Dibenzo-p_-dioxin was synthesized according to the procedure described by
Gilman and Dietrich (14). 1-Chloro- and 2-chlorodibenzo-p_-dioxin were pre-
pared by the method of Pohland and Yang (24). 2,3-Dichloro- and 2,7-dichloro-
dibenzo-£-dioxin were obtained from RFR Corp. 2,8-Dichloro- and 1,2,4-tri-
10
-------
chlorodibenzo-p_-dioxin were obtained from the Food and Drug Administration.
2-Hydroxydibsnzo-£-dioxin was synthesized by a rr-thcd analogous to that des-
cribed by Hartoucjh and Keisel (15) for the synthesis of 2-hydroxydibenzothio-
phsne. Pyrocatechol was obtained from Eastman Kodak, Inc. 1,2-DihycJroxynaph-
thalene VMS synthesized by the method of Corner and Your,;-; (6). 2,3-Oihycroxy-
biphenyl was prepared ?>s previously described (13). Oxidized nicotind.iiiJe
adenine dinuclcotide (;IAu^) was obtained from Sir^s Clinical Co. Si lice-Gel
60 was from Brinkman Instruments, Inc. Basic Almina, Brocl-.n^n Activity I was
from Fisher Scientific Co. BioRad AG-1X8 was obtained from EioRad Laborator-
ies, Pi ethyl amincethyl (DEAE)-cellulose w?s obtained from Whatman. Inc. Ana-
lytical t.l.c. was performed using Brinkman Chromatogram sheets, type Sil G/UV
(Silica-Gel with fluorescent indicator). Preparative t.l.c. was conducted
with glass plates (5.0 x 20.0 cm) precoated with Silica-Gel 60 (Brink-en In-
struments, Inc.). Organic solvents were purified by vacuum distillation. All
other materials were of the highest purity commercially available and were
used without further purification.
11
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SECTION 5
RESULTS AND DISCUSSIONS
Preliminary, Studies
The initial approach was to attempt to isolate new microorganisms with
the ability to degrade dibenzo-p_-dioxin. The biodegradation of this compound
would be studied in detail with respect to metabolites formed as well as the
effect of the oxidation on the organism. However, numerous attempts to iso-
late microorganisms from freshwater and a wastewater treatment facility, capa-
ble of utilizing dibenzo-£-dioxin as a sole carbon source proved unsuccessful.
In a subsequent experiment, several different strains of bacteria, pre-
viously shown to be capable of oxidizing aromatic hydrocarbons, were screened
for the ability to co-oxidize dibenzo-£-dioxin. Organisms were grown in 100
ml cultures of mineral salts broth with 0.2% succinate as the carbon source.
Dibenzo-£-dioxin (0.05%) was added to the cultures as a solid. Growth of the
experimental cultures was compared to the growth of control cultures grown on
succinate (0.2%) in the absence of dibenzo-p_-dioxin. The formation of products
in the culture medium was analyzed qualitatively by thin-layer chromatography
as described in Materials and Methods. Results are summarized in Table 1.
Dibenzo-£-dioxin was unable to support the growth of any of the organisms when
supplied as the sole carbon source. However, co-oxidation was observed under
certain condtions. Among the strains tested, a Pseudomonas sp. and a Beijer-
inckia sp. were selected for further studies.
12
-------
TABLE 1. SUGARY OF THE PRELIMINARY SCREENING OF ORGANISMS
FOR THE ABILITY TO CO-OXIDIZE DIDEf.'ZO-^-DIOXIN
Organism
Growth
t'etdbolite Forr-;tion
J Color F.eectic
Beijerinckia sp.
Beijerinckia sp. strain B8/35
+
4-HH- 0.22
0.5
None detected
Brov:n
Purple
Pseudo-^mas sp. N.C.I.B. 9816
Pseudomonas sp. N.C.I.B. 9816
strain 11
£.- P'Jtid,-' NP
P_. putida NP strain 119
P.. putida TOL
P. putida TOL strain 39/D
Flavobacterium sp.
Mycobacterium sp.
Acinetobacterium sp.
-H--H-
•H-++
ilone detco'c^d
0.5 (trace) Fainl purple
None detected
None detected
None detected
None detected
0.5 (trace) Faint purple
None detected
None detected
^
Chfomatograms developed in CHCl3:acetcne (SO/20)
Color reaction with Gibbs reagent and subsequent exposure to ammonia vapors
Oxidation of Various Aromatic Hydrocarbons by Esajjaaitl^s. sp. N.C.I.S. 9816
Duplicate cultures (500 ml) of Pseudomonas sp. N.C.I.B. 9816 were grown
to mid-log phase on the various carbon sources listed in Table 2. Oxygen con-
sumption bv washed, whole cells of Pseudomonas sp. N.C.I.B. 9816 was then ex-
amined in the presence of various aromatic substrates (Table 2). Cultures
grown with succinate or on succinate with an inducing substrate exhibited
little activity with the various substrates. Alternatively, cultures grown
13
-------
TABLE 2. OXIDATION OF SUBSTRATES BY WASHED, WHOLE CELLS OF PSEUDOMONAS SP. N.C.I.B. 9816
Oxygen uptake on
Growth Substrate(s)
0.2% Succinate
0.2% Succinate +
0.5% Salicylate
0.2% Succinate +
0.05% Naphthalene
0.2% Succinate +
0.05% Dibenzo-£-dioxin
0.1% Salicylate
0.1% Naphthalene
Naphthalene
27.6
23.2
19.2
9.1
294.0
263.3
various
Benzene
5.6
4.9
5.8
0.0
21.1
24.6
substrates (nsr.oles of 02 consumed/min ing )
Anthracene
6.8
6.3
5.2
0.0
36.8
46.7
Dibsnzo-p-diox1n
6.4
8.3
8.9
0.0
21.1
24.6
Catechol
0.0
4.3
4.8
0.0
215.4
183.8
Enzyrcatic activity was monitored polarographically as described in Materials and Methods.
Aromatic substrates were added in N.N-dimethylformamide (200 nmoles/10 pi).
-------
with salicylate or naphthalene as the sole carbon source readily oxidized the
various aromatic substrates. No attempt was made to isolate and identify the
metabolites.
Oxjdaticn of dibenzQ-.n-dioxin by Pseudo^ionas sn. N.C.I.B. 9816 strr.in 11
Preliminary studies of the oxidation of dibenzo-g_-dioxin by Pseudr.'onas
sp. N.C.I.B. 9816 strain 11 indicated that co-oxiduticn \:as negligible v;hen
the organism was grown with succinite (0.21") as the carbon source. However,
when Pseudomonas sp. N.C.I.B. 9816 strain 11 was grown in 500 ml cultures of
mineral salts broth containing 0.1% salicylate and 0.05% dibenzo-2.-dic.xin, two
neutral products (compounds A and B) were excreted into the culture rcsdium (Fig. 1)
Compound A (Rf 0.22) absorbed ultraviolet light and gave a brown color with
Gibbs reagent. Compound B (Rf 0.5) also absorbed ultraviolet light and gave
a purple color with Gibbs reagent. The formation of compound A in the culture
medium was monitored spectrophotometrically (Figure 2). Maximum accumulation
of compound A was observed after 36 hours.
It is of interest that Pseudomonas sp. N.C.I.B. 9816 strain 11 failed to co-
oxidize dibenzo~2-dioxin when grown on succinate but readily oxidized the com-
pound when grown on salicylate. This observation is consistent with the re-
port by ShamsuzzamanandBarnsley (28) suggesting that in this organism, sali-
cylate is the coordinate inducer of a block of enzymes catalyzing reactions
in the conversion of naphthalene to salicylate. The results also indicate
that dibenzo-p_-dioxin fails io induce the enzymes responsible for the oxida-
tion of the aromatic nucleus.
Isolation of Compounds A and B
Pseudomonas sp. N.C.I.B. 9816 strain 11 was grown in 10 liters of mineral
salts broth containing 0.1% salicylate and 0.05% dibenzo-p_-dioxin. After 36
15
-------
front
4
80:20
O O-
o
B
-g»
.1 p-
3 A
Figure 1. Thin-layer chromatogram of the products formed from
the oxidation of dibenzo-p_-dioxin by Pseudomonas sp.
N.C.I.B. 9816 strain 11. Experimental details are
discussed in the text.
16
-------
9CO
i.^\j
Wove I e not h (nm)
Figure 2. Formation of compound A by Pse:idomonas sp. N.C.I.B. 9816 strain 11
(legend next page).
hours, the culture was filtered to remote unused dibsnzo-o-dioxin and then ex-
tracted with an equa' volume of ethyl acetate. The organic liyer was dried
over ani.ydrous Na2SO^ and the solvent removed in vacuo at 35 C to yield 367 mg
of a solid residue.
The residue was dissolved in a small volume of chloroform and applied to
the top of a silica-(jel column (1.9 x 22.0 cm; 25% ^0 w/v). Thp column was
eluted with 700 mis of chloroform and then 700 mis of chloroform/rrethanol
(98:2). The presence of compounds A and B in the colurr.n fractions (15 mis)
was detected by thin-layer chroruatography. Fractions 9-15 were found to con-
tain compound B. Compound A was eluted in fractions 63-75. Frscf-ons con-
17
-------
Figure 2 Legend.
A 500 ml culture was grov/n as described in the text. At the times indicated
(hr rs) the accumulation of compound A was measured quantitatively as described
in Materials and Methods.
-------
taining each metabolite were pooled *nd the solvent removed in. vf»cno at 20 C.
Compound A (242 rcg) end compound B (276 rcg) were further purified by two re-
crystallizations from hexene/acetonc.
Identification of Compound B
The white crystals obtained after rccryr-tallizution h-d the following
physical properties: raiting point, 1<:*-K5 C; nass spectrum, calculated for
12ci2llH31G°3' 200-0473' found m-ss. 200.0*68. The infrared snctnr- (K-jjo",;
exhibited raxirca at 2.62, 2.92, 5.15, 6.32, 7.53, 10.83, 12.5, ar.d 13.4i.m
(Figure 3). The ultraviolet spectrum (KeOH) showed maxima at 221 n~ (c =
32000) and 292 nm (e = 4300) (Figure 4). The proton magnetic resonance spec-
trum of compound B in deuterated acetone is sho-.=n in Figure 5. An analysis of
the spectrum is given in Table 3.
4000 03CO 2GOO 1SSO
o.oi"~T'"";''' ' ' "•' '•
.10
,.n:-s
OA-1 KX50 TO? r.30 7C3
• . J__ _i*.__ . * * J ,i_t * ti^fA* j* 4 j f a j*A*>.* ji^AAii. + -- * * i • • ' -
f?30
p
^.50
-H-
-^0
-j.20
-.20
.40
.50
1.0
1.0
i3 12
Figure 3. Infrared soectruni of compound B (fiujol)
15
18
-------
£20 £40 ££O 2&Q
'WAVELENGTH
SOO
520
Figure 4. Ultraviolet spectrum of compound B (HaOH)
The results of the physical data suggest that compound B is 2-hydroxydi-
benzo-p_-dioxifi. Verification of this structure was obtained with the chemi-
cal synthesis of 2-hydroxydibenzo-p_-dioxin from 2-bromodibenzo-p_-dicxin via
an alkali fusion reaction. Compound B exhibited infrared, ultraviolet and
mass spectra identical to those of the synthetic compound. No depression of
nelting point was observed when compound B was mixed with the authentic sample
of 2-hydroxydibenzo-p_-dioxin.
19
-------
II
Figure 5. Proton magnetic resonance spectrum of compound B
20
-------
TABLE 3. ANALYSIS OF THE PROTON MAGNETIC RESONANCE SPECTRUM CF COMPOUND B
(3)H
OH (4)
K(3)
it (2)
Prnton'
;
2
3
4
Chenfc&t Shift (&}
6.77
6.46
6.B2-7.02
8. 20-8.44
Description
IK [9, croaatic;
2H {a, aroaetlc]
-------
Identification of Compound A
The white crystals obtained after recrystallization had the following
physical properties: malting point, 139-140 C; [a]*5 + 110° (c - 0.405, MeOH);
mass spectrum, calculated for 12C12lH1016°4> 218.0579, found mass, 218.0537.
The infrared spectrum (Nujol) showed peaks at 3.05, 5.92, 5.3, 6.9, 7.95,
10.82, 12.0, 12.4, and 14.13 u:n (Figure 6). The ultraviolet spectrum in
methanol (Figure 7) exhibited maxima et 227 rim (c 12500} cr.ci 298 r.m (c =
2800). The proton magnetic resonance spectrum of compound A in deutcratcd
pyridine is shown in Figure 8. An analysis of the spsctrim is riven in Table
4.
4000 3COO 2CCO i:00 C.V IOCS reo CCS 7C3
fc-^^fc* 1.4 1.1. ' <^ I,) I I Ut.- ii'.n. J-J iiii. i '• ' I t. i I. - U^^-^J^M-i' 1 I I, ' I I I I ' >->.l ,^J-4-J—^^_l_« . i,
.1C
U.20-
o'""L^"j^|^;
f ' ' " "
-h^—'.20
^_ _J_:l4._;
^ —i-.-i—::o
Figure 6. Infrared spectrum of compound A (Nujol)
The physical data suggest that compound A is 1,2-dihydroxy-l,2-dihydro-
dibenzo-p_-dioxin. Additional support for the assigned structure was provided
by the tcid-catalyzed dehydration of compound A. Compound A (53 mg) was dis-
solved in 20 ml of ethyl ether. Concentrated HC1 (5.0 ul) was added to the
solution. After 1 hour of intubation at 27 C the solvent was removed.
22
-------
leaving 45 nig of a solid residue. The physical properties of the resulting
compound were found to be identical with compound B which was previously iden-
tified as 2-hydroxydibGnzo-£-dioxin.
O 0.8
tt.
cc
O
0.4
0.2
220 2<0 ?.CO 260 500
WAVELENGTH (ntn)
320
Figure 7. Ultraviolet spectrum of compound A (MeOH)
23
-------
Figure 8. Proton magnetic resonance spectrum of compound A (Pyridine-d^).
The hydroxyl protons were exchanged with D£0 before recording
the spectrum as they interferred v/ith the signal given by H(5).
-------
TABLE 4. ANALYSIS OF THE PROTON MAGNETIC RESONANCE SPECTRUM OF COMPOUND A
H{5)
(5JH
K(5)
Proton Cheaical Shift (i)
1 4.55
2 4.S2
3 5. SO
4 6. 06
5 6.70-7.00
i
IK {dt hydroxy^ithlnc,
4j'g • 6.5 H»J
1H fa, hydreisyasthine,
Jj ^ = C.S Hz]
1H (lid. ok-flnit,
Jj « » tQ.O Hi
Jj^ - 3.0 Hz)
1H (a, oleflnlc.
J<|3 • 10.0 Hz]
4H (CD, aronvsttc)
25
-------
Although the results obtained establish the structure of compound A as
l,2-dihydroxy-l,2-dihydrodibenzo-p_-d-ioxin, they do not distinguish between
the possible cis and tr?ns stereoisomers. Evidence for a cis configuration
was obtained by the synthesis of an isopropylidine derivative (3). Compound
A (290 tng) was suspended in 20 ml of 2,2-dinethoxypropana. The suspension
was chilled in an ice bath prior to the addition of 1.0 rcg of p_-toluenesul -
fonic ecid. The reaction was monitored by thin-layer chromatcKraphy. After
4 hours the presence of compound A in the reaction mixture could no longer be
detected, however, a nonpolar compound (compound C; Rf 0.64) was observed
that absorbed ultraviolet light and gave a yellow-brown color with Gibbs
reagent. At this point, triethy la-nine (0.2 ml) was added to neutralize the
acid and the solvent was removed in vacuo at 35 C. The oily residue (335 ng)
was dissolved in a small volume of hexane and applied to the top of a column
of basic alumina (1.0 x 16.0 cm). Elution with benzene/hexane (1:1) yielded
90 mg of a white solid. Compound C had the following physical properties:
melting point, 97-99 C; [a]5 + 232° (c = 0 41 in methanol ) ; mass spectrum,
calculated for 12C151H141604, 253.0892, found mass 253.0885. The infrared
spectrum (Nujol) showed maxima at 5.85, 6.27, 6.93, 7.93, 7.95, 8.67, 8.9,
10.8, 12.7, and 13.4 ym (Figure 9). The ultraviolet spectrum in methanol
(Figure 10) exhibited absorption maxima at 228 nm (e = 13900) and 297 nm (e =
2300). The proton magnetic resonance spectrum is shown in Figure 11. An
analysis of the proton magnetic resonance spectrum of compound C is given in
Table 5. These observations establish the structure of compound C as 1,3-
di oxol 0-2 ,2-dimethyl -1 ,2-di hytirodi benzo-p_-di oxi n .
26
-------
4CDO 3DCO 2COO J5C3
0.0^r
.10
ICCO 900 CSO
S-
<
£.20
: t V ''
re
> T t a iit LJ^-i , k H* ij yiiin.t - -j fca i n.'-NJ ^jin^.- ••irfiiali'S^'-
U 12
Figure 9, Infrared spectrum of compound C (Nujol)
The present study has demonstrated that cis-1,2-dihydroxv-l.2-dihvdrodi-
benzo-£-dioxin is the first detectable metabolite in the oxidation of dibenzo-
£-dioxin by a Pseudomonas sp. It should be noted that the cis-stereochemis-
try of the !:/droxyl groups was inferred by the formation of an isopropylidene
derivative witn 2,2-din^thoxypropane (3) and also from the fact that cis-
dihydrodiols have been implicated as intermediates in the bacterial metabolism
of a variety of aromatic hydrocarbons (11). The cls-dibenzo-£-d1oxin dihydro-
diol can subsequently undergo acid-catalyzed dehydration to yield 2-hydroxy-
dibenzo-£-dioxin.
-------
240 LJO 2L3 SCO
WAVELENGTH (nrr.J
Figure 10. Ultraviolet spectrum of compound C (HeOH)
28
-------
II
LJ
Figure 11. Proton magnetic resonance spectrum of compound C (CDC10)
29
-------
TABLE 5. ANALYSIS OF TiiE PF.OTOFi fJlSuETIC RESOilfJiCE SfECTRi;; OF CC::"Ci:;!D C
<6)H
K{5)
FrotxJB
Chdtcal Shift {«)
Descrtplisn
1.44
«.90
s.ea
6.62-6.50
3H l». KttrylJ
El (S.
IK Id.
J. - • 3.0
1H
J, , • 6.0 i:z
j^': • 3.0 lu)
1H ((M. olcHntc.
J . r • 10.0 Hi
J*1* • 3.0 Hll
\H Id. clfflntc.
J, . • 10.0 Hi)
-------
Metabolism of T,2-d1hydroxy-7,2-dihydrodibenzo-,n-dioxiri by cell extracts
When cell extracts prepared from the parent organism v;ere incubated under
anaerobic conditions with cis-1,2-dihydroxy-l,2-dihydrodibenzo-p-dioxin and
NAD+, a product of the reaction (compound D) s*as detected by thin-layer chro-
matography (Rf 0.3") that absorbed ultraviolet light and gave an intense brown
color with Gibbs reagent. In a large-scale reaction, crude cell extract (80
mg of protein) was mixed with ci_s_-l ,2-dlhydroxy-l ,2-dihydrodibenzo-p_~dicxin
(0.7 mmoles) and NAD+ (1.0 mmoles) in 40 ml of 0.05 H KH2P04 buffer, pH 7.2,
and incubated under anaerobic conditions in a sealed serum bottle. After 3
hours, the reaction mixture was acidified with 0.5 ml of 5 N HC1 and extracted
with three volumes of ethyl acetate. The organic extracts were pooled, dried
over anhydrous f^SO^, and the solvent removed in vacuo at 35 C to yield 185
mg of a solid residue. The residue was suspended in a small volume of chloro-
form and loaded onto the top of a silica-gel column (1.0 x 20.0 cm; 25% HjC).
The column was eluted with 500 mis of chloroform and 10 ml fractions were
collected. The presence of compound D TT fractions 16-28 was detected by thin-
layer chrosnatography. These fractions were pooled and the solvent rernoved to
yield 90 mg of compound D which was further purified by two recrystallizations
from hexane/acetone.
Identification of Compound D
The white crystals obtained after recrystallization had the following
physical properties: melting point, 172-173 C; mass spectrum, calculated for
12c12lH816o4' 216-0422, found mass, 216.0422. The infrared spectrum (Nujol)
showed maxima at 2.85, 3.06, 6.15, 6.3, 7.73, 10.8, 12.72, and 13.4 vm (Fig-
ure 12). The ultraviolet spectrum in methanol (Figure 13) exhibited maxira
at 229 nm (c = 30800) and 289 ran (c = 3300). The proton magnetic resonance
31
-------
spectr-m of compound D in douterated acetone is sho,;n in Figure 14. An ?naly-
sis of the spectrun is given in Table 6. Due to a rapid excr.or?? rate the hy-
droxyl protons were not observed at arrbient temperature, hov.-ever they ware
observed at 8.65 6 when the spectrum was record-! at -15 C.
The results of the physical data surest that cc7pc»nd D is l ,2-dihydroxy-
dibcnzo-£-dioxin. This observation is consistent i.-;th t^? f. ct thn several
£is_-dihydrodiols have been shown to be oxidized to cct?crois tv pyridine
nucleotide-d,?pendent dehydrooenases (1,23,25,26).
4
.._.
,-T
3000
-•
::.
:::..;-
• -
-^r-
/T-j-
Mi.
H
i:
2C
-
•
CO 1500 OV 1(^0 9^0 f'.D /CO
'* I \~~ ''-'I • i " T ---' — 1 - -| : * - ' •' i * T
• — r~ -.—• ' k— • ' 1 — .- - •
: • . :
~~
--)--;-. -- -r '--- ^"-, -^ L— . ^:f-^- .:- •-!--•' ; ;- •[ \ '-;
i •-;• r---" ' • • i • " ' • ! : 1 • • • - i ' '. • ' :
•• . . !!' -1 : ! -I . li i . i--!-.;. • ; ' '
l ' ' ' 1 ' " " ; ' • i 1 - t i ' ' ' '
• •
::::;:::-j:::-: . • -j -.;.{•.--) J-;.;- : i" i j t ; <
........... . 1 . .. , - , 1 1 . . . 1 . ,
L '
...., .
... :.. ....
- ' • ' • ' r- . -•! . l i - i '. : i . : . v
' : ' ' ! '. ' j '• ':.[.-' i I ' ' |
lllll . .;!. ..li.,L
^iiilu^ UW ii.liii ?". ' r' ' :- ' -- K ..a !_j 1.... ...i .... U_l
--3.0
— j.;o
-
l
'"
rn
. . , 7n
— jl.O
— <70
7 C 9 ID it i2 13 J4 li,
WAVHIWGTH (Ar^CCO-M-
Figure 12. Infrared spectrtzn of compound D (Nujol)
-------
240
ECO 200 £00
WAVELENGTH (on)
Figure 13. Ultraviolet spectrum of coiripound D (MeOH)
33
-------
I
JL
Figure 14. Proton magnetic resonance spectrum of compound D (Acetcne-d^)
Oxidation of 1 ,2-dihydroxydibonzo-p-riioxin by cell extracts
Polarographic experiments were performed to test the ability of 1,2-
dihydroxy-dibenzo-p_-dioxin to serve as a substrate for the 1,2-dihydroxynaph-
thalene oxygenase in extracts of the parent strain of Pseudorr.jnas sp. fi.C.I.B.
9816 (Figure 15). An initial investigation revealed that cell extracts were
extremely active on the physiological substrate 1,2-dihydroxynaphthalene (4724
nmoles of 02 consumed/min per mg of protein), however only slight activity was
observed with 1,2-dihydroxydibenzo-p_-dioxin (54 nmoles of C^ consureo'/min per
mg of protein). It was also noted that incubation of cell extracts (C.15 rcg
of protein) with as low as 10 nmoles of 1 ,2-dihydroxydibenzo-p_-dioxin led to
the complete inhibition or inactivation of the 1,2-dil)ydroxyr,3?lithclene oxygen-
ase, as additions of 1.0 pmole of 1,2-dihydroxynaphthalene did not result in
further enzyrratic activity.
34
-------
TABLE 6. ANALYSIS OF THE PROTON MAGNETIC RESONANCE SPECTRUM OF COMPOUND D
HC3)
OH (4)
(3}H
(3)H
OHM)'
H{3)
HC2)
Proton
Chcaiwl Shift («}
tescrJptlon
6.45
6.52
S.D3-7.08
8.25-6.S6
1H [de
o.o (i
JH [d. arcaatlc
J, , • 2.0 H
4H (•. fi row tie)
2H Ib, hydroxylj
-------
100.0
s
3
cr>
1 c
o
o
O 50.0r
vt
o
0.0
0.5 1.0
Time (min)
Figure 15. Polarographic assay of 1 ,2-dihydroxynaohthalene oxycenase in
cell-free extracts of Pseudcrconas sp. N.C.I.B. 9S15.
Oxidotion of dibenzo-p-dioxin and chlorinated dibgnzo-p-dioxins by Pfii
sp.
Duplicate cultures (500 ml) of the parent strain of Sei jerinckia sp. were
g,x>wn to mid-log phase in mineral salts broth on succinate with biphenyl as an
inducing substrate. In addition, an anion-exchanqe resin (AG 1-X8; 5.0 g/1)
was included to remove acid products (19). The ability of whole cells of
Bei jerinckia sp. to oxidize dibenzo-£-dioxin and several chlorinated dibcnzo-
pj-dioxins was investigated. The rate of oxidation of the dibenzo-p-dioxins
was compared to the ability of the organism to oxidize bipiienyl. Results of
35
-------
.-. '
-<
Figure 15 Legend.
Polarographic assay of 1,2-dihydroxynaphthalene oxygenase in cell-free
extracts of Pseudoinonas sp. N.C.I.B. 9816.
Activity of the 1,2-dihydroxynaphthalene oxygenase was monitored polaro-
graphically as described in Materials and Methods. A protein concentra-
tion of 0.15 mg was used in the analysis. The reaction was initiated
witn the addition of; 100 nrnoles of 1,2-dihydroxynaphthalene in 10 ul
of anhydrous tetrahydrofuran ( ), 10 nmoles of 1,2-dih3'droxy-
dibenzo-p_-dioxin in 10 yl of anhydrous tetrahydrofuran ( ), and
100 nmoles of 1,2-dihydroxydibenzo-p-dioxin in 10 pi of anhydrous tetrs-
hydrofuran ( ). At the point indicated by the arrow 1.0 vimole
of 1,2-dihydroxynaphthalene (in 10 pi of anhydrous tetrahydrofuran) was
added. All rates were corrected for endogenous respiration and for the
nonenzymatic oxidation of 1,2-dihydroxynaphthalene. No auto-oxidation
of 1,2-dihydrcxydibenzo-p_-dioxin was observed under the assay conditions
described.
-------
the ^investigation are summarized in Table 7. Dibenzo-2.-dioxin wes oxidized
at a lower rate than the monochlorinated diber.zo-^-dioxins. The fact that
monochlorinated dibenzo-£-dioxins were oxidized faster than the parent com-
pound may reflect substrate or product inhibition of the enzy:r.?s systems in-
volved. Further studies are needed to clarify thi: point. The rate of oxi-
dation of the chlorinated dibenzo-o-dioxins oc-creared with an incrriring de-
gree of chlorine substitution.
TABLE 7. OXIDATION OF SUBSTRATES BY WASHED, WHOLE CELLS OF BEToZRr'^'r SP.
Substrate1
Siphenyl
Dibenzo-p_-dioxin
l-Chlorodibenzo-p_-dioxin
2-Chlorodibenzo-p_-dioxin
2,3-Dichlorodibenzo-p_-dioxin
2,7-Dichlorodibenzo-p^-dioxin
2,8-Dichlorodibenzo-£-dioxin
l,2,4-Trichlorodibenzo-£-dioxin
Specific activity^
72.3
29.8
52.5
52.6
11.2
8.5
8.5
6.5
Relative rate (x')
100.0
41.2
72.6
72.7
15.5
11.3
11.8
8.9
^All substrates added in N,N-dimethylfonnamide (200 nmole5,/10.0 vl)
^Oxygen uptake at 30 C (nmoles of Oo consumed/mi n per nv} of protein)
Oxidation of dibenzo-D-dioxin (I) by B^jerindiia. B3/35
When BeijrHnckia B8/36 was grown in mineral salts madiun containing 0.2?
succinate and 0.05* dibenzo-p-dioxin (I) a neutral product {compound 1A) was
37
-------
detected in the culture medium. Compound IA (Rf 0.22) absorbed ultraviolet
light and gave a brown color with Gibbs reagent. Growth of the culture and
accumulation of compound IA with time are shown in Figure 16. After 24 hours,
duplicate cultures were extracted to leave 5.8 mg of a solid residue. Purifi-
cation of the residue led to the isolation of 3.0 mg of a crystalline product
that melted at 135-136 C (compound IA). The metabolite had mass, infrared
and ultraviolet spectra identical to those of 1,2-dihydroxy-l,2-dihydrodibenzo-
£-dioxin. This product was previously obtained from the co-oxidation of di-
benzo-p_-dioxin by Pseudomonas sp. N.C.I.B. 9816 strain 11. Further support
for this structure was provided by the conversion of compound IA to 2-hydroxy-
dibenzo-p_-dioxin. Compound IA (2.0 mg) was suspended in 3.0 ml of methanol
and the reaction was initiated with the addition of 10.0 pi of 5.0 N ^SO^.
After 30 min incubation at 27 C the solvent was removed, leaving 2.0 mg of a
solid residue. Thin-layer chromatography of the residue revealed that com-
pound IA was converted to a phenolic product (compound IB; Rf 0.5) that geve
a purple color with Gibbs reagent. The latter compound was also detected in
the co-oxidation cultures after 20 h. Compound IB was identified as 2-hydroxy-
dibenzo-p_-dioxin, having a melting point, mass, infrared and ultraviolet spec-
tra identical to those of the known standard. Preliminary evidence for a cis-
configuration of compound IA (1.0 mg) was suspended in 3.0 ml of 2,2-dimethoxy-
propane and the reaction was initiated with the addition of 0.1 mg of p_-tolu-
enesulfonic acid. Analysis of the reaction mixture by thin-layer chrornatog-
raphy revealed the formation of a nonpolar product (compound 1C; Rf 0.64) that
absorbed ultraviolet light and gave a yellow-brown color with Gibbs reagent.
These results indicate that c^s_-l,2-dihydroxy-l,2-dihydrodibenzo-n-dioxin is
the first detectable metabolite of the oxidation of dibenzo-p_-dioxin by
33
-------
-'"•'•'e SP- This compound can subsequently undergo a r;cn-cr..'ry-3tic dehy-
dration to yield 2-hydroxydiben2C-£_-dioxin.
1.2
1,0
« °'S
o
c
I O'S
t/>
.a
<
0,4
0,2
0,0
4
Tims (h)
Figure 16. Growth and accumulation of compound IA by cultures of
Beijerinckla E3/35. (legend next page).
Oxidation of chlorinated dibenzo-n-dioxins by B^lisrj_rc,kj? DS/j^
Beijcrinckia 38/36 was grov.n in duplicate cultures as above with the ex-
ception that dibenzo-jv-dioxin was replaced by 1-chloro- (II), 2-chloro- (III),
39
-------
Figure 16 Legend.
A 500 ml culture was grown as described in the text. Growth of the culture
was monitored turbidometrically at 600 nm ( ). Accumulation of
compound IA was measured quantitatively as described., in Materials and
Methods. Analysis of the ultraviolet spectrum of compound IA indicated
an absorption maximum at 227 nm. Accumulation of compound IA with time
is expressed as A227 per ml of culture ( ).
-------
2,3-dichloro- (IV), and 2,7-dichloro- (V) dibenzo-n-dioxins. The accu-,:ul?.tion
of neutral products (compounds IIA and IIIA) was detected in cultures contain-
ing monochlorinated dibenzo-P_-dioxins (Figure 17). Ho products wsre cetected
in cultures grown in the presence of two dichlorinated dibenzo-p-dioxins.
Cultures containing monochlorinated dibpnzo-£_-dioxins v:ere extracted with
ethyl acetate. Subsequent purification led to the isolation of 2.5 nq end 1.5
mg of compounds IIA and IIIA, respectively. The properties of the crystalline
products are surmarized in Table 8. The results indicate tfv.t the monochlor-
inated dibenzo-£-dioxins were oxidized to di'nydrodiols. These results also
indicate that dibenzo-p_-dioxin and monochlorinated dibenzo-o_-dioxins incuce
the synthesis of enzymes responsible for oxidation of the aromatic n-jclius in
the Beijerinckia sp. Furthermore, it appears that the dichlorinatcd dibenzo-
p_-dioxins fail to function as inducing substrates. Thus, the degree of chlor-
ine substitution on the aromatic ring may have an effect on the ability of a
compound to induce the synthesis of degradative enzymes in this organism. In
contrast, previous studies have demonstrated that dibenzo-p-dioxin failed to
induce the synthesis of enzyitri necessary for aromatic hydrocarbon degradation
in Pseudomonas sp. N.C.I.B. .-^ifc.
Further support for the formation of dihydrodiols was indicated by the
acid-catalyzed dehydration of compounds IIA and IIIA to phenols (Figure IS).
Compound IIA was converted to a product (compound IIB; Rf 0.5) that absorbed
ultraviolet light and gave a purpla color with Gibbs reagent. Compound IIIA
was also converted to a phenolic product (compound IIIB; Rf 0.51) that ab-
sorbed ultraviolet light and cave a dark blue color with Gibbs reagent. Ad-
ditional products were not detected in the reactions mixtures. It is of in-
terest that the acid-catalyzed dehydration of the dibenzo-p_-dioxin and the
-------
8 12 16 20 24 28
Time (h)
Figure 17. Growth and accumulation of compounds IIA and IIIA by
cultures of Beijerinckia B8/35. ('legend next page).
monochlorinateci dibenzo-p_-dioxin dihydrodiols appears to result in the forma-
tion of single phenolic products. This phenomenon may be due to resonance
stabilization or conformation of the dehydration intermediates and hence may
favor the formation of single phenolic products (2,17).
41
-------
Figure 17 Legend.
Cultures (500 ml) containing 1-chloro- (II) and ?-ch1orcdibon:o-2.-
dioxin (III) were grown as described in the text. Grov/th of the cul-
tures was monitored turbidometrically at 600 nm ( ). Accu-
mulation of compounds IIA and IIIA w?s measured quantitatively as
described in Materials and Methods. Analysis of the ultraviolet
spectrum of compound IIA indicated an absorption maximum at 235 m.
Compound IIIA exhibited an absorption maximum at 237 nm. The accu-
mulation of compounds IIA and IIIA with time is expressed as fylb
( ) and A^37 ( ) per ml of culture, respectively.
I '\
-------
TABLE 8. PHYSICAL PROPERTIES OF THE fitTABOLITES FORMED FROM MONOCHLGRIMATED
DIBEHZO-£-DIOXIHS BY EEIJCRIKCKIA B8/36.
Property
Rf (chloroforro:acetone)
Gibbs reaction
Melting point
U.v. maxima (methanol)
I.r. maxima (nujol )
Mass spectra (m/e)
Number of Cl
Proposed structure
IIA
0.21
Brown
130-131 C
235 ran
303 nm
3.10 urn
5.95 pm
6.37 urn
9.45 VJHI
252 (M+)
234 (-H20)
1
C12H904C1
Compound
IIIA
0.21
Brown
n.d.1
237 nm
309 PIT:
3.07 urn
5.57 vm
6.37 urn
9.45 pm
252 (H+)
234 (-H?0)
1
C-j2^qOfl^
1
Not done
Preliminary evidence for a c_i£-stereochemistry of the hydroxyl groups of
the dihydrodiols (corrpounds IIA and IIIA) was obtained with the syntheses of
isopropylidins derivatives. Compounds IIA and IIIA were converted to nonpolar
products, compounds IIC (Rf 0.62) and IIIC (Rf 0.62), respectively, when incu-
bated in the presence of 2,2-dimethcxypropane as described above. Both of the
latter products absorbed ultraviolet light and gave tan colors with Gibbs re-
42
-------
T
ACID
Compound IIA —-» 110
2 20
300
\Vave!cngth (nm)
340
o
c
O
XI
s,
O
O
.Q
IIIO
c.;
c G
0.2
0.0
ACID
Compound MIA —> IM3
220
,.j „,' '_-„<
\Vcv3lcngth (nm)
Figure 18. Acid-catolyrrrd deh>rfrat1oT of compounds IIA and IJIA
(legend next page)
-------
Figure 18 Legend.
Ultraviolet absorption spectra of compounds IIA and IIA was rror.itored
before (solid lines) and sfter (dashed lines) acidification with H-,50^
as described in Materials and .fethoris. Each compound was isolated and
purified by preparative thin-layer chrorratography and recrystallized
from hexane-acetone. Compounds IIA and IIIA (1.0 mg) were dissolved in
3.0 ml of methanol and the ultraviolet spectrum recorded. Dehydration
of compounds IIA and IIIA was initiated with the addition of 10.0 ul of
5.0 N !I2S04 and the solutions were incubated at 27 C for 30 min. Reac-
tions were monitored by thin-layer chromatography as described. Upon
completion of the acid-catalyzed dehydration the ultraviolet spectrum
was again recorded.
J fr
-------
agent. The isopropylidene derivatives of compounds IIA and IIIA wer3 round
to be highly unstable and decomposed to products resembling compounds IIC end
IIIB. Thase result^ indicate that os_-dihydrodiols are also ths initial \r.-j-
tabolites from the oxidation of rr.onochlorinated dibenzo-p-dioxins by Brn_er-
Inckjj^ sp.
Oxidation of dibenzo-n-dioxin (I) by ppjjprir.rki^ qn.. ui 1 d tvn?
When the parent strain of BeylGrirckjia \:as grown in ninorai r^lts r.:eir-r
containing 0.2% succinata and 0.052 dibenzo-p_-dioxin (I), no products v?rc- de-
tected by thin-layer chromatography or spectrophctometric analysis of th-_ cul-
ture medium. In addition, the growth of the organism v/as inhibited after 4
hours (Figure 19). To determine if further oxidation was possible, a 1er.;c-
scale culture of Beijerinckia sp. was grown as described with 0.2" succir.jt?
and 0.05" biphenyl. The washed cell pellet (5.0 g wet wt) was resusr?r:ce.d in
500 ml of 0.05 M KH2P04 buffer, pH 7.2, containing 0.05% dibenzo-n-dioxin.
The resting cell suspension was incubated on a rotary shaker at 27 C. After
12 hours, the suspension was filtered through glass wool to remove mused
dibenzo-p_-dioxin. The filtrate was adjusted to ph 4.0 with concentrated H-^O.;
and extracted with two volumes of ethyl acetate. The organic layer was dried
over anhydrous Na^SO/j and the solvent removed, leaving 21.0 mg of an oily resi-
due. Analysis of the residue by thin-layer chroratography revealed the forma-
tion of a neutral product (compound ID; Rf 0.3) that absorbed ultraviolet
light and gave an intense brown color with Gibbs reagent. Purification of ths
residue led to the isolation of 6.0 mg of a crystalline product that melted
at 172-173 C. Compound ID was identified as 1,2-dihydroxydibenzo-p_-dioxin,
having mass, infrared and ultraviolet spectra identical to those of the bio-
logical product previously characterized.
-------
a
.n
t-
o
0,5
0,4
>.2
0.1
0,0
12
Time (h)
i >••»
lo
o/i
Figure 19. Effect of dibenzo-£-dioxin on the growth of
Beijerinckia sp., wild type
It is of interest that growth of the parent strain of Beijerinckia on
succinite was inhibited when dibenzo-p_-dioxin (0.05%) was included in the cul-
ture medium. As cis-T ,2-dihydroxy-l,2-dihydrodibenzo-p-dioxin was not inhibi-
tory for the growth of the mutant strain (B8/36) of Beijerinckia, the toxic
effects may be due to the oxidation of dibenzo-p-dioxin to 1,2-dihydroxydiben-
zo-£-dioxin. The toxicity of 1,2-dihydroxydibenzo-p_-dicxin was inferred from
from the fact that the compound was found to accumulate when resting cell sus-
pensions of the parent strain of Dgijorinckia were incubated in the presence
45
-------
Figure 19 Legend.
Cultures (500 ml) of the parent strain of Ooi.icrincki^ Here gro.vn in min-
eral salts broth with 0.2% succinate as the carbon source. E/.p"-ir?ntc/i
cultures contained 0.052 dibenzo-£.-dioxin. Growth of control ( 'l
and experimental ( —) cultures was rcor.itortd turbidcrnetr:cully
at 600 nm.
-------
of dibenzo-j[>-dioxin. Furthermore, growth inhibition of the parent strain was
observed after four hours. This period corresponds approximately to the amount
of time required by the organism for induction of enzymes necessary for aro-
matic hydrocarbon oxidation, as indicated by the accumulation of c_js_-l ,2-di-
hydroxy-1,2-dihydroc*ibenzo-£-dioxin after four hours by Beijerinckia B3/36.
Oxidation of1;2-dihyciroxydibenzo-p.-dioxin by cell extracts
Polarographic experiments with cell extracts were conducted to test, the
ability of 1,2-dihydrox.ydibenzo-p_-dioxin to serve as a substrate for enzymes
responsible for the oxidative fission of the aromatic nucleus (Figure 20).
100
o
S
c
a>
6
1 50
o
w
o"
0.5 1.0
Tims (mtn)
5,5
Figure 20. Polarographic assay of ring-fission oxygenases in
cell extracts of Beijer1ncM_a sp. (legend next page)
46
-------
Figure 20 Legend.
The reaction vessel contained, in a tot.il volume of 1.4 ml, 0.05 !•; ^i'?.;
buffer, pH 7.5, and cell extract (0.81 mg of protein). The reaction \:as
initiated with the addition of 100 nmoles of catechol ( ). 1 r,--,u.-«
of 2,3-dihydroxybiphenyl ( ), 100 nmoles of 1 ,2--dihydroxyditcri::c.-£_-
dioxin ( ) and 10 nmoles of 1,2-dihydroxydibenzo-g_-ciioxin ( },
all in 10 vl of N,N-dirr>ethy1forrcamide. At the time indicated by the arro.'
( + ), 100 nmoles of catechol or 2,3-dihydroxybiphenyl was odd-;d. All rite-
were corrected for endogenous respiration. No auto-oxidation of tho c^t.e-
chol substrates was observed under the assay conditions described.
-------
An initial investigation revealed the ce'Jl extracts were active on the physio-
logical substrates, 2,3-dihydroxybiphenyl (64.1 nir.oles of Q2 consumed/min per
mg of protein} and catechol (212.2 nmoles of 02 consumed/min per mig of pro-
tein). Cell extracts exhibited a brief initial rate of oxidation of 1,2-
dihydroxydibenzo-£-dioxin (98.6 nmoles of 02 consumed/min per mg of pvotein)
and then rapidly fell to the non-enzymatic rate. In addition, incubation of
cell extracts (0.81 mg of protein) with as low as 10 nmoles of 1,2-dihydro>:y-
dibenzo-£-dioxin led to complete inhibition or inactivation of the catechol
and 2,3-dihydroxybiphenyl oxygenases, as additions of 100 nmoles of either
substrate did not result in further enzymatic activity.
To determine if both enzymes catalyzing the oxidative fission of the aro-
matic nucleus were capable of oxidizing 1,2-dihydroxydibenzo-g-dioxins the
2,3-dihydroxybiphenyl oxygenase was separated from the catechol oxygenase by
ion-exchange c;iromatography (Figure 21). A crude cell extract (0.23 g of pro-
tein) was loaded onto a column of DEAE-cellulose (1.4 x 8.0 cm) which had been
previously equilibrated with 0.05 M KH2P04 buffer, pH 7.5, containing 10%
acetone (phosphate-acetone buffer). The column was washed with 50 ml of
phosphate-acetone buffer, and 10 ml fractions were collected. The two activi-
ties were eluted separately using a stepwise KC1 gradient in phosphate-acetone
buffer. 2,3-Dihydroxybiphenyl oxygenase was eluted with 0.2 M KC1 in phos-
phate-acetone buffer (fractions 18-21) and exhibited activity with both 2,3-
dihydroxybiphenyl and ce-techol. The catechol oxygenase was eluted with 0.4 M
KC1 in phosphate-acetone buffer (fractions 23-28). Fractions from each peak
with the highest specific activity were pooled and utilized in further studies.
To determine if tho activity present in fractions 18-21 was due to a
single protein species, the effect on activity of incubation at 55 C with
-------
0.0
Frcstlen
Figure 21. Separation of the 2,3-dihydroxybiphenyl and catechol
oxygenases by ion-exchange (DEAE) chromatography
(legend next page)
time was investigated. The activity in the fraction for both 2,3-dihydroxy-
biphenyl and catechol was found to decrease at the same rate with tine. Thus,
it was assumed that the activities were due to the action of a single enzyne,
which was tentatively identified as the 2,3-dihydroxybiphenyl oxygensse.
Polarographic experiments were performed to test the ability nf 1,2-
dihydroxydibenzo-p_-dioxin to serve as a substrate for the partially purified
48
-------
Figure 21 Legend.
Protein was eluted from the column with a stepvn'se gradient of 60 ml of
0.1 M KC1 in phosphate-acetone buffer, 70 ml of 0.2 M KC1 in phosphate-
acetone buffer and 120 ml of 0.4 M KC1 in phosphate-acetone buffer.
Samples (10-100 yl) of each fraction were analyzed spectrophotometrically
for oxygenase activity with catechol U - &) and 2,3-dihydroxybipher.yl
(a - a) as described. Enzyme activity is expressed as moles of product
fcrmed/min per ml of fraction. Protein concentration"(e - ©) in the frac-
tions was determined by the method of Lowry e_t al_. (20).
-------
2,3-dihydroxybiphenyl and catechol oxygenases. The results of the £n:lyses
are surcnarlzed in Table 9. The 2,3-dihydroxybiphenyl oxyganas- (O.-S r-? Of
protein) was capable of oxidizing both 1,2-dihydroxydibenzo-P-dioxin ir.t 2,3-
dihydroxybiphenyl, while the catcchol oxyganase (0.54 n.g of protein) \;?G un-
able to oxidize 1,2-dihydroxydibenzc-p_-dio>:in.
Spectrophotcmetric assays were utilized for kinetic analyses to c;t>rrr,ire
the type of inhibition exhibited by 1,2-dihydroxydibenzo-p_-dioxin \/ith re^r^ct
to the physiological substrates of the partially purified 2,3-dih;^roxyhio!ic-,-;yi
and catechol oxygenases. All inhibition studies were conducted after r,-0-inrL.
bation of the enzyme with the inhibitor for 1 min prior to initinticn of tht
reaction with the appropriate substrate. Activity of the 2,3-dihyoro. • ji-
phenyl oxygenase was monitored using a protein concentration of 0.12 r.-.'n1
The Michaelis constant (Km) for 2,3-dihydroxybiphenyl was 8.3 pM. The ^',r.-
of varying concentrations of 2,3-dihydroxybiphenyl was determined in t1-? •:'.-:•:-
ence of 1.0 pM 1,2-dihydroxydibeni. g-dioxin. Results of the ana'yscs v:-rc
used to prepare a Lineweaver-Burke plot (Figure 22a). Act'.vity of the <;L~>. -
chol oxygenase v/as monitored using a protein concentration of 0.2 r.g/nl. "me
Km for catechol was found to be 7.1 yM. The effect of varying concentration?.
of catechol was determined in the presence of 0.4 pM 1,2-dihydroxydi?er^o-r-
dioxin. A Lineweaver-Burke plot was prepared from the resu'its (Figure 22b).
These investigations indicate that 1,2-dihydroxydibenzo-p_-dioxin is a potent
mixed-type inhibitor of both the 2,3-dihydroxybiphenyl and catechol oxyqc-nasc-:,
present in this organism.
It is noteworthy that the further Jegradation of 1,2-dihydroxyaiber.zo-^-
dioxin does not appear to be favored due to the inhibition and possibly t:>e
inactivation of the ring-fission dioxygenases by this metcral ite. A similar
49
-------
TABLE 9. POLAROGRAPHIC ANALYSIS OF THE ACTIVITIES OF THE PARTIALLY
PURIFIED 2,3-DIHYDROXYBIPHENYL AND CATECHOL OXYGENASES.
2,3-Dihydroxybiphenyl oxyqenase
Substrate1
2,3-D-" hydroxybi phenyl
1,2-Dihydroxydibenzo-£-dioxin
Specific activity^
35.6
64.43
Catechol oxyqenase
Substrate Specific activity
Pyrocatechol 206.9
1,2-Dihydroxydibenzo-p_-dioxin 0.0
TAH substrates added in N.N-dimethylformamide (100 nmoles/10.0
Specific activity (nmoles of Q£ consumed/min per rng of protein)
Initial rate - fell rapidly to non-enzymatic level
phenomenon was observed during the oxidation of dibenzo-p_-dioxin by Pseudomnnas
sp. N.C.I.B. 9816. The present investigation nas revealed that 1,2-dihydroxy-
dibenzo-p_-dioxin is a potent mixed-type inhibitor of both the 2,3-dihydroxy-
bi phenyl and catechol oxygenases present in the Beijerinckia sp. The nature
of this inhibitory phenomenon is under current investigation.
50
-------
I L^-l I—.1 !><)(«,
SO 14
-g- x toj(r.r()
Figure 22. E.'fcct of L 2-dihydroxydib-jr:;o-p_-dTO.x-in or m
-------
Figure 21. Effect of 1,2-dihydroxydibenzo-p_-dioxin on ring-fission
oxygenases from Bei.ierinckia sp.
A. Effect of 1,2-dihydroxydiben70-p.-dioxin on 2.3-dihvdrox.ybiphenyl
oxygenase.
The enzyme was pre-incubated in the presence of 1.0 yH 1,2-dinydroxy-
dibenzo-p_-dioxin for 1 min prior to initiation of the reaction with
substrate. Activity of the 2,3-dihydroxybiphenyl oxygcnase was de-
termined at various concentrations of 2,3-dihydroxybiphenyl in t::;
presence (o o) and absence (e ©) of the inhibitor.
B. Effect of 1,2-dihydroxydibgnzo-p-dioxin on catechol oxyngnase.
The enzyme was pre-incubsted in the presence of 0.4 pM 1,2-dihydroxy-
dibenzo-p_-dioxin for 1 min prior to initiation of the reactk.n with
substrate. Activity of the catechol oxygenase was determined at, vari-
ous concentrations of catechol in the presence (o o) and absence
(a e) of the inhibitor.
52
-------
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-------
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