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Applied and Environmental Microbiology, October 2000, p. 4247-4252, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Substrate Specificity of Atrazine Chlorohydrolase
and Atrazine-Catabolizing Bacteria
Jennifer L.
Seffernick,1,2
Gilbert
Johnson,1
Michael J.
Sadowsky,2,3,4 and
Lawrence P.
Wackett1,2,3,*
Department of Biochemistry, Molecular
Biology, and Biophysics,1 Biological
Process Technology Institute,3 Center
for Microbial and Plant Genomics,2 and
Department of Soil, Water, and Climate,4
University of Minnesota, St. Paul, Minnesota 55108
Received 20 April 2000/Accepted 17 July 2000
 |
ABSTRACT |
Bacterial atrazine catabolism is initiated by the enzyme atrazine
chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by
investigating the catalytic activity of AtzA by using substrate
analogs. Purified AtzA from Pseudomonas sp. strain ADP
catalyzed the hydrolysis of an atrazine analog that was substituted at
the chlorine substituent by fluorine. AtzA did not catalyze the
hydrolysis of atrazine analogs containing the pseudohalide azido,
methoxy, and cyano groups or thiomethyl and amino groups. Atrazine
analogs with a chlorine substituent at carbon 2 and N-alkyl
groups, ranging in size from methyl to t-butyl, all
underwent dechlorination by AtzA. AtzA catalyzed hydrolytic
dechlorination when one nitrogen substituent was alkylated and the
other was a free amino group. However, when both amino groups were
unalkylated, no reaction occurred. Cell extracts were prepared from
five strains capable of atrazine dechlorination and known to contain
atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain
PATR, Alcaligenes strain SG1, Agrobacterium
radiobacter J14a, and Ralstonia picketti D. All
showed identical substrate specificity to purified AtzA from
Pseudomonas sp. strain ADP. Cell extracts from
Clavibacter michiganensis ATZ1, which also contains a gene
homologous to atzA, were able to transform atrazine analogs
containing pseudohalide and thiomethyl groups, in addition to the
substrates used by AtzA from Pseudomonas sp. strain ADP.
This suggests that either (i) another enzyme(s) is present which
confers the broader substrate range or (ii) the AtzA itself has a
broader substrate range.
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INTRODUCTION |
The s-triazine ring is
present in many compounds used to make dyes, resins, and herbicides
(15, 19, 29). Herbicides are directly applied to soils or
plants, and their environmental longevity is largely dependent on
bacterial degradation. Many s-triazines were initially
thought to be poorly degraded, but more recent studies indicate that
they are completely mineralized by a number of bacterial isolates
(9, 21, 25). For instance, in 1937, melamine
(2,4,6-triamino-1,3,5-s-triazine) was reported to be
nonbiodegradable. In 1964, however, bacteria capable of slow
degradation were isolated. More recently, melamine was reported to be
readily biodegraded (8). In a similar manner, bacteria capable of catabolizing the s-triazine herbicide atrazine
(2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) to carbon
dioxide were not reported during the first 30 years of application to
agricultural fields. Atrazine is the most heavily used
s-triazine herbicide in the United States, with about
68 × 106 to 73 × 106 lb applied to
fields each year (17, 20, 35, 36). Identification of
numerous atrazine-degrading bacteria in the last decade suggests that
bacteria have evolved new degradative abilities due to exposure to atrazine.
Initially, bacteria capable of dealkylation and deamination of atrazine
and atrazine analogs were isolated (4, 15, 22-24, 26, 33).
A cytochrome P-450 in Rhodococcus sp. strains TE1 and
N186/21 was shown to remove the alkyl substituents of atrazine via an
oxygenative mechanism (26, 33, 37). Triazine hydrolase (TrzA) from Rhodococcus corallinus NRRL B-15444R catalyzes
the deamination of unalkylated aminotriazines, such as melamine or 2-chloro-4,6-diamino-1,3,5-s-triazine (CAAT). TrzA catalyzes
a slow dechlorination reaction with desethylsimazine (CEAT) but fails
to catalyze either deamination or dechlorination reactions with
atrazine (9). Other bacteria and consortia with the ability to dechlorinate less highly substituted triazines like desethylatrazine were also identified; however, these bacteria were unable to
dechlorinate atrazine (4, 9).
Prior to 1993, there was no knowledge of a bacterial catabolic pathway
for atrazine that proceeded via an initial dechlorination reaction
(20, 30, 39). Shortly thereafter, a number of bacteria with
atrazine dechlorination activity were isolated (1, 7, 11, 30,
36; K. Boundy-Mills, unpublished data).
Pseudomonas sp. strain ADP was one of the first bacteria
shown to metabolize atrazine to carbon dioxide, ammonia, and chloride.
In this bacterium, the atrazine metabolic pathway proceeds via
three consecutive hydrolytic reactions, which remove the chloride,
N-ethylamine, and N-isopropylamine substituents,
thereby converting atrazine to cyanuric acid. The enzymes that catalyze
these reactions are atrazine chlorohydrolase (AtzA), hydroxyatrazine
ethylaminohydrolase (AtzB), and isopropylammelide
isopropylaminohydrolase (AtzC), respectively (6, 14, 31).
All three enzymes were identified as members of the amidohydrolase
protein superfamily by computer-based sequence analysis
(31).
Other bacteria capable of atrazine dechlorination have been
independently isolated from different locations around the world under
a variety of isolation conditions: (i) Rhizobium strain PATR
(7), (ii) Alcaligenes strain SG1 (Boundy-Mills,
unpublished), (iii) Agrobacterium radiobacter J14a
(36), (iv) Ralstonia picketti D (M. L. de
Souza, N. R. Plechacek, L. P. Wackett, M. J. Sadowsky, and B. L. Hoyle, Abstr. 98th Gen. Meet. Am. Soc. Microbiol. 1998, abstr. 453, 1998), and (v) Clavibacter michiganensis ATZ1
(1, 11). Data from PCR and Southern hybridization
experiments indicate that all of these bacteria contain genes
homologous to those that are active in the atrazine degradation pathway
of Pseudomonas sp. strain ADP. Moreover, the sequence of an
internal 0.5-kb region of the atzA gene from each of the
bacteria was more than 99% identical to that of atzA from
Pseudomonas sp. strain ADP (13).
Previous studies suggest that strains containing homologs to the
Pseudomonas sp. strain ADP atzA have slightly
different triazine degradation profiles (7, 11, 14, 36).
Purified AtzA from Escherichia coli DH5
(pMD4) containing
the atzA gene from Pseudomonas sp. strain ADP was
reported to degrade atrazine and simazine but not melamine,
terbuthylazine, or CAAT (14). Cell-free lysates from
Rhizobium strain PATR were shown by thin-layer
chromatography (TLC) to degrade atrazine, simazine, and terbuthylazine
but not propazine or melamine (7). Consequently, these
investigators concluded that two isopropyl groups prevented catalysis.
A. radiobacter J14a was assayed for degradation of atrazine,
ametryn, cyanazine, prometon, simazine, and propazine in culture media
with whole cells. High-pressure liquid chromatography (HPLC) analysis
showed that 62 to 100% of all triazines tested were degraded in
120 h (36). C. michiganensis ATZ1 was shown
to degrade ametryn, prometryn, simazine, propazine, simetryn, and
atratone in a whole-cell consortium with Pseudomonas strain
CN1 (11). Due to different assay conditions and detection
methods, the results described in the literature are difficult to
compare with the substrate specificity of purified AtzA.
In this study, in vitro investigations were conducted to determine the
substrate specificity of enzymes initiating the metabolism of atrazine
and analogous compounds. First, purified AtzA from Pseudomonas sp. strain ADP was incubated with atrazine
analogs. To expand the study, we synthesized and assayed numerous
s-triazine and pyrimidine compounds that are not
commercially available. Subsequently, in vitro enzymatic cell extracts
prepared from other atrazine-degrading bacteria were investigated for
their ability to degrade the atrazine analogs and the degradation was
compared to degradation by AtzA from Pseudomonas sp. strain ADP.
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MATERIALS AND METHODS |
Bacterial strains and protein purification.
Pseudomonas sp. strain ADP, Rhizobium strain
PATR, Alcaligenes strain SG1, A. radiobacter
J14a, C. michiganensis ATZ1, and R. picketti D
were grown in R minimal medium (13) at 30°C with 100 µg
of atrazine per ml as the sole nitrogen source. Cells were harvested by
centrifugation at 15,000 × g for 2 min and resuspended in 25 mM morpholinepropanesulfonic acid (MOPS) (pH 7). Crude cell extracts were prepared by lysing cell suspensions via three freeze-thaw cycles followed by sonication three times at 80% intensity for 20 s each, using a Biosonik sonicator (Bronwill Scientific, Rochester, N.Y.). Cell debris was removed by centrifugation at 15,000 × g for 15 min.
Purified AtzA protein was isolated from E. coli DH5(pMD4)
(14). Cultures were grown at 37°C overnight in
Luria-Bertani medium (32) supplemented with chloramphenicol
(30 µg/ml). The cells were lysed and AtzA was purified as previously
described (12).
Atrazine analogs.
Atrazine, simazine, propazine,
terbuthylazine, ametryn, prometryn, desethylatrazine,
desisopropylatrazine,
2-chloro-4-hydroxy-6-(N-isopropylamino)-1,3,5-s-triazine (CIOT), 2-chloro-4-(N-ethylamino)-6-hydroxy-1,3,5-s-triazine (CEOT), N-isopropylammelide, ammeline, and ammelide were
graciously provided by Novartis Crop Protection (Greensboro, N.C.).
Commercially available CAAT was purchased from Sigma Chemical Co. (St.
Louis, Mo.). All other compounds were synthesized specifically for this study as described below (Table 1).
All triazines and pyrimidines synthesized for this study were analyzed
by gas chromatography mass spectrometry (GC-MS) using
an HP 6890/5973
instrument (Hewlett-Packard, San Fernando, Calif.)
(Table
1). Compounds
were purified by crystallization and silica
gel chromatography. Purity
was confirmed by GC, HPLC, or TLC
analyses.
The 2-substituted atrazine analogs were prepared through substitution
reactions which replaced the chloride substituent of
atrazine.
Fluoroatrazine and cyanoatrazine were prepared by heating
atrazine with
excess potassium fluoride or cyanide in dimethyl
sulfoxide at 140°C
or in dimethylformamide at 120°C, respectively
(
16,
27).
Products were characterized by GC-MS and
1H and
13C nuclear magnetic resonance spectroscopy. A similar
procedure
was used to produce azidoatrazine from the sodium azide salt,
with a lithium bromide catalyst in methyl ethyl ketone at 80°C.
Aminoatrazine was prepared by hydrogenation of azidoatrazine in
isopropanol with a 5% palladium-on-calcium-carbonate catalyst
under a
hydrogen atmosphere. Treatment of atrazine with excess
sodium
methoxide in methanol at room temperature yielded atratone
(methoxyatrazine).
Production of dichloro-mono-
N-alkylamino triazine
intermediates were essential for the synthesis of various
N-alkylamino triazines.
The intermediates were formed by
reacting cyanuric chloride
(2,4,6-trichloro-1,3,5-
s-triazine)
(Aldrich Chemical Co.,
Milwaukee, Wis.) with alkyl amines or ammonia
in dichloromethane or
anhydrous diethyl ether at 0 to 5°C. The
reaction of the dichloro
intermediates with alkylamine or ammonia
in ethanol was used to aminate
the triazine ring a second time,
thereby producing
chloro-di-
N,
N'-alkylaminotriazines. Alternately,
a cold 10% aqueous solution of ethylamine, methylamine, or ammonia
was
treated with the dichloro intermediate in acetone or tetrahydrofuran
to
produce the desired
chloro-di-
N,
N'-alkylaminotriazine. By altering
the alkylamines used,
N-methyldesisopropylatrazine,
N-methylsimazine,
and
N,
N'-dimethylsimazine were
prepared.
Preparation of the dichloro-mono-
N-alkylaminopyrimidine
intermediate resembled that of the dichlorotriazine intermediate,
using 2,4,6-trichloropyrimidine (Aldrich Chemical Co.) as
starting
material, except that the reaction mixtures were maintained at
room temperature. The 4,6-dichloro-2-alkylaminopyrimidine and
2,6-dichloro-4-alkylaminopyrimidine isomers formed in roughly
equal
proportions and were separated by crystallization. As with
the
triazines, a second alkylamino substituent was added to the
dichloro
intermediate in ethanol. Gentle heating was required
for addition of
t-butylamine. The compounds
2-chloro-di(
N-ethylamino)-1,3-pyrimidine
(CEEP),
2,6-di(
N-ethylamino)-4-chloro-1,3-pyrimidine (ECEP),
2,6-di(
N-isopropylamino)-4-chloro-1,3-pyrimidine
(ICIP),
2-chloro-4-(
N-ethylamino)-6-(
N-isopropylamino)-1,3-pyrimidine
(CIEP),
2-chloro-4-(
N-
t-butylamino)-6-(
N-ethylamino)-1,3-pyrimidine
[C(tB)EP],
2-(
N-
t-butyl amino)-4-chloro-6-
N-(ethylamino)-1,3-pyrimidine
[(tB)ECP], and 2-(
N-ethylamino)-4-chloro-6-(
N-
t-butylamino)-1,3-pyrimidine
[E(tB)CP] were prepared in this
manner.
Substrate incubation and analysis.
Saturated solutions of
the various triazine and pyrimidine compounds listed in Table 1 were
prepared in 10 mM phosphate buffer (pH 7.0). Due to the low solubility
of most triazines, saturated solutions provided adequate concentrations
for detection but prevented the determination of kinetic constants. The
triazine and pyrimidine solutions were incubated with 50 µl of cell
extracts for 16 and 48 h at 30°C. Enzymatic reactions were
stopped by heating for 2 min at 95 to 100°C. Control samples without
enzyme were handled in parallel with the enzyme-treated samples.
Samples were analyzed by HPLC, using an Hewlett-Packard HP 1100 system
equipped with a photodiode array detector interfaced to an HP
ChemStation. An Adsorbosphere C18 5µ column (250 by 4.6 mm) (Alltech, Deerfield, Ill.) was used to separate alkylated triazines
and pyrimidines with an acetonitrile-water linear gradient as
previously described (6). Unalkylated substrates were
separated on an Alltech Inertsil 5µ phenyl column (150 by 4.6 mm)
with an isocratic aqueous mobile phase consisting of 5 mM sodium octane
sulfate in 0.05% H3PO4.
Two colorimetric assays were developed to monitor fluoride production
in enzymatic reactions with fluoroatrazine as a potential
substrate.
The first was modified from the procedure of Bellack
and Schouboe
(
5), using 500-µl samples. In this method, fluoride
disruption of a red complex formed by
sodium-2-(p-sulfophenylazo)-1,8-sihydronephthalene-3,6-disulfonate
(SPANDS) and zirconium(IV) ions results in a bleaching of color.
Standard curves had an 8 to 80 µM linear range. The second method
was
an alizarin-lanthanum assay modified from Sigma Protocols
(Sigma
Chemical Co.) for 800-µl sample sizes. This method had
a 10 to 130 µM linear
range.
Two independent procedures were used to monitor ammonia production in
reactions, using aminoatrazine as a potential substrate.
The first used
the Berthelot method as described by Okamura and
Kigasawa
(
28) and Weatherburn (
38) with modifications for
50-µl sample sizes. This assay incorporates the nitrogen from
ammonia, through a series of chemical reactions, into indophenol,
which
produces a blue color. Standard curves had a 10 µM to 10
mM linear
range. The second method used an enzymatic assay kit
(no. 171-A)
developed by Sigma Chemical Co. The reductive amination
of
2-oxoglutarate to glutamate by glutamate dehydrogenase occurs
with the
concurrent oxidation of NADPH to NADP
+, which was monitored
at 340
nm.
| |
RESULTS AND DISCUSSION |
Purified AtzA substrate specificity.
Substrate analogs were
synthesized to investigate the catalytic activity of purified AtzA from
Pseudomonas sp. strain ADP (Table 1). Previously, only
atrazine and simazine had been shown to be substrates. In this study,
AtzA was shown to displace the fluoride substituent of fluoroatrazine.
Fluoride release was also monitored via two spectroscopic methods.
However, the acidic conditions required for chromophore formation
hydrolyzed fluoroatrazine in the absence of enzyme, thereby preventing
an accurate quantification of enzyme-catalyzed fluoride release. The
rate of fluoroatrazine conversion to hydroxyatrazine by AtzA was 108% ± 9% (n = 3) of that of enzymatic atrazine
dechlorination at equivalent substrate concentrations of 50 µM. The
Vmax of AtzA with fluoroatrazine could not be
determined due to the low water solubility of the substrate. However,
the observation of statistically similar rates with either fluoride or
chloride substituents at equimolar concentrations suggests that the
carbon-halogen bond energy does not strongly influence the rate of the
AtzA-catalyzed reaction. Bromoatrazine was synthesized, but the high
rate of spontaneous hydrolysis in water precluded reliable enzyme assays.
Atrazine analogs containing azido, cyano, methoxy, or thiomethyl groups
in place of the chlorine substituent of atrazine were
investigated with
purified AtzA from
Pseudomonas sp. strain ADP.
Both analog
disappearance and potential formation of hydroxyatrazine
were monitored
by HPLC. No activity was detected by either criteria.
Although AtzA can
hydrolytically remove halides, it did not remove
pseudohalides (Fig.
1A). Pseudohalides, like azido, methoxy,
and
cyano groups, have a similar size, electronegativity, and
reactivity
in substitution reactions to halides, but they are composed
of
multiple atoms. The thiomethyl group is common to the herbicides
ametryn and prometryn. AtzA from
Pseudomonas sp. strain ADP
catalyzed
the dechlorination of atrazine and propazine but did not
remove
the thiomethyl group of the structurally analogous ametryn and
prometryn.
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FIG. 1.
Substrate analogs of atrazine. (A) Substituents
hydrolyzed by various atrazine-dechlorinating strains. (B)
Chlorodialkylamino triazines determined to be dechlorinated by cell
extracts of Pseudomonas sp. strain ADP, Rhizobium
strain PATR, Alcaligenes strain SG1, A. radiobacter J14a, R. picketti D, and C. michiganensis ATZ1.
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AtzA from
Pseudomonas sp. strain ADP also failed to remove
the free amino group from aminoatrazine. Assays for hydroxyatrazine
and
ammonia were both negative. Aminoatrazine is of particular
interest
because AtzA has been identified as being evolutionarily
related to
enzymes catalyzing deamination reactions (
31). Sequence
alignments of AtzA with enzymes such as urease and adenosine deaminase
have revealed it to be a member of the amidohydrolase protein
superfamily (
18,
31). It has been known since the 1960s that
members of this family are also capable of dechlorinating substrate
analogs. Adenosine deaminase, for instance, catalyzes deamination
and
dechlorination reactions of analogous purine substrates (
2,
3,
10). The deamination and dechlorination reactions of
s-triazine
hydrolase (TrzA), however, are not
performed on the same substrates.
TrzA catalyzes the deamination
of diaminotriazines and pyrimidines
and the dechlorination of
triazines which have a single alkyl
group on one of the two nitrogen
substituents, with deamination
reactions favored over dechlorination
reactions (
34). The inability
of AtzA to catalyze
deamination reactions with aminoatrazine,
as well as other
aminotriazines and pyrimidines such as melamine
and CAAT, places AtzA
in a novel dehalogenating subgroup of the
amidohydrolase
superfamily.
Since numerous members of the amidohydrolase protein superfamily are
involved in purine or pyrimidine metabolism, pyrimidine
analogs were
also synthesized and tested as potential substrates
for AtzA.
Pyrimidine analogs of atrazine, simazine, terbuthylazine,
and propazine
[CIEP, CEEP, ECEP, C(tB)ep, (tB)ECP, and E(tB)CP]
were prepared with
a chlorine substituent in the 2 and 4 positions.
None of the pyrimidine
substrates tested were substrates for
AtzA.
The structural requirements of AtzA with respect to the
N-alkyl side chains were also investigated.
Mono-
N-alkylated triazines
with one of the nitrogen side
chains alkylated and the other unalkylated
(e.g., desethyl and
desisopropylatrazine) underwent dechlorination
by AtzA. One of the
nitrogens on the triazine ring could therefore
be unsubstituted, but if
both were unsubstituted, as with CAAT,
hydrolytic dechlorination was
not observed. The requirement of
at least one
N-alkyl group
on the triazine ring is consistent
with the alkyl group being
significant in substrate positioning
and
orientation.
The sizes of the alkyl groups on each of the nitrogen substituents were
investigated and appeared to be less significant (Fig.
1B). AtzA
catalyzed dechlorination with triazine ring substrates
containing
N-methyl, ethyl, isopropyl, and
t-butyl
substituents.
Compounds with groups larger than
t-butyl were
too insoluble for
analysis. Simazine, propazine,
terbuthylazine,
2-chloro-4,6-
N,
N'-dimethyl-1,3,5-
s-triazine,
and
2-chloro-4,6-
N,
N'-ditertbutyl-1,3,5-
s-triazine
were substrates
for AtzA from
Pseudomonas sp. strain ADP.
Compounds containing
tertiary amino side chains like
N-methyl-deisopropylatrazine [C(EM)AT],
N,
N'-dimethylsimazine, and
N-methylsimazine were not substrates.
These triazines
containing tertiary amino substituents were most
probably not
substrates, either due to size and orientation restrictions
or due to
the absence of an amino hydrogen. The amino hydrogen
has the potential
to hydrogen bond in the enzyme active site,
thereby assisting in
orientation or generating an alternate configuration
of electrons in
the triazine ring during the dechlorination reaction.
The ability of
AtzA to dechlorinate compounds with secondary amino
side chains which
are larger than some of the tertiary amino side
chains suggests that
electronic factors are potentially more significant
than steric
factors.
Enzymatic activity of cell extracts from atrazine-degrading
strains.
Similar assay conditions were used to compare the
substrate degradation profiles of six atrazine-dechlorinating strains.
For all of the compounds tested (Table 1), cell extracts of
Pseudomonas sp. strain ADP, Rhizobium strain
PATR, Alcaligenes strain SG1, A. radiobacter
J14a, and R. picketti D had identical substrate degradation
profiles to that described for purified AtzA from Pseudomonas sp. strain ADP. Previously, each of those
strains were shown by PCR to contain an atzA gene with a
sequence nearly identical (>99% identity) to the atzA gene
from Pseudomonas sp. strain ADP (13). By
contrast, cell extracts from C. michiganensis ATZ1 were able
to degrade ametryn, prometryn, atratone, cyanoatrazine, and
azidoatrazine in addition to the substrates degraded by the other strains (Fig. 1A).
Pseudomonas sp. strain ADP was reported previously not to
degrade terbuthylazine (
12). However, in this study,
terbuthylazine
was shown to be a substrate for AtzA from
Pseudomonas sp. strain
ADP. A previous report indicates that
Rhizobium strain PATR does
not degrade propazine
(
7); however, propazine degradation was
observed in this
study. The different observations could be because
propazine is
extremely insoluble in aqueous solution. Therefore,
the 1-h incubation
time and relative insensitivity of the TLC
assay in the previous study
may have precluded the observation
of propazine degradation. Another
apparent difference between
the results presented here and those in the
literature involves
A. radiobacter J14a. In the present
study, transformation of triazine-containing
methoxy and thiomethyl
groups was not observed. Previously,
A. radiobacter J14a was
reported to degrade ametryn, cyanazine, and
prometon. Prometon has a
methoxy group similar to atratone. The
different observations could be
due to methodological differences;
the previous study was done with
whole cells, while this study
was conducted using cell extracts.
Although AtzA is soluble and
active in cell extracts, membrane-bound
enzymes or enzymes that
require additional cofactors or cosubstrates
might not be active
in the soluble cell extracts used here. There is
precedence for
a cytochrome P-450 being able to carry out atrazine
dealkylation
(
4,
15,
22-24,
26,
33). Dealkylated products
of atrazine
were detected in whole-cell studies, suggesting that other
enzymes
might also be involved in atrazine degradation (
36).
Previously,
de Souza et al. examined
C. michiganensis
ATZ1, in a consortium
with
Pseudomonas strain CN1, for
degradation of
s-triazine substrates
(
11).
Whole-cell assays revealed that the consortium degraded
methoxy and
thiomethyl substrates. However, whether this degradation
was due to
dealkylation or actual hydrolysis of the substituent
in the 2 position
of the triazine ring was not determined. In
our study, we provide the
first evidence that
C. michiganensis ATZ1 is capable of this
activity and that hydroxyatrazine is the
first metabolite produced.
C. michiganensis ATZ1 degraded ametryn,
prometryn, atratone,
cyanoatrazine, and azidoatrazine in addition
to the substrates
degraded by the other strains. Whether this
additional activity is due
to activity of an AtzA homolog with
broader substrate specificity or to
additional enzymes is being
investigated.
The specific activity of the atrazine dechlorination reaction for each
of the six strains was determined (Table
2). The specific
activity of
Pseudomonas sp. strain ADP was nearly twice those
of other
strains. Differences between the strains could either
be due to
differences in the protein ratio of the AtzA homolog
protein to other
proteins in the cell or be due to differences
in the activity of the
enzyme itself. The data presented here
do not distinguish between these
two cases.
C. michiganensis ATZ1
had an extremely low
specific activity for atrazine dechlorination
(0.05 ± 0.02 nmol/min/mg). The specific activity for the conversion
of ametryn to
hydroxyatrazine is 0.10 ± 0.03 nmol/min/mg. Although
the
difference in rates with these two substrates is not large,
ametryn was
repeatedly degraded more quickly than atrazine. Plates
containing
concentrations of ametryn above the saturation limit
also consistently
showed clearing zones before plates containing
atrazine. The increased
rate of the conversion of ametryn to hydroxyatrazine
over the
conversion of atrazine to hydroxyatrazine suggests that
the strain
is better suited for ametryn degradation than for atrazine
degradation.
| |
ACKNOWLEDGMENTS |
We thank the following researchers for providing strains: David
Crowley and coworkers for C. michiganensis ATZ1, T. B. Moorman for A. radiobacter J14a, Kyria Bounty-Mills for
Alcaligenes strain SG1, P. Plesiat and C. Bouquard for
Rhizobium strain PATR, and Blythe Hoyle and Nathan Pechacek
for R. picketti D. Rhodococcus corallinus NRRL
B-15444R was obtained from the National Center for Agriculture
Utilization Research in Peoria, Ill., with permission of Walter Mulbry.
Special acknowledgment of the contributions of Jack Richman should be
noted for his initial synthesis of cyanoatrazine and for helpful
discussion. We also thank Carol Somody, Janis McFarland, and Andrea
Elder of Novartis Crop Protection for providing s-triazine
compounds and metabolites.
This research was supported, in part, by a grant from Novartis Crop
Protection and by NIH training grant GM08347.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Molecular Biology, and Biophysics, Biological Process
Technology Institute, 1479 Gortner Ave., University of Minnesota, St.
Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-1700. E-mail: wackett{at}biosci.cbs.umn.edu.
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Abstract
Introduction
