Enzymatic Degradation of Chlorodiamino-s-Triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazinebiodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzesthe dechlorination of atrazine but is unreactive with CAAT.In this study, melamine deaminase (TriA), which is 98% identicalto AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine(CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase(AtzB) to yield ammelide. This represents a newly discovereddechlorination reaction for AtzB. Ammelide was subsequentlyhydrolyzed by N-isopropylammelide isopropylaminohydrolase toproduce cyanuric acid, a compound metabolized by a variety ofsoil bacteria.

INTRODUCTION

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Introduction

s-Triazine herbicides, such as atrazine and simazine, commonlyundergo oxidative dealkylation reactions in soils. Soil fungi(14, 15, 17) and Rhodococcus strains (3, 19) have been shownto catalyze dealkylation reactions with s-triazine substrates.In the Rhodococcus strains, a cytochrome P450 monooxygenaseenzyme (ThcB) catalyzes a single dealkylation reaction (3, 19).However, double dealkylated metabolites, such as 2-chloro-4,6-diamino-s-triazine(CAAT), have been detected in soils and with pure cultures offungi (13, 17). Klebsiella pneumoniae strain AZ was isolatedfor its ability to grow with CAAT as a nitrogen source, butthe enzymatic basis for this metabolism was not defined in thator subsequent studies (9).

In contrast, the bacterial metabolism of atrazine to carbondioxide and ammonia is well defined and known to occur via aseries of hydrolytic reactions. For example, atrazine chlorohydrolase(AtzA) from Pseudomonas sp. strain ADP catalyzes the hydrolyticdechlorination of N-alkylated s-triazines, such as atrazineand simazine (Fig. 1A) (6). Subsequent hydrolytic deaminationreactions are catalyzed by hydroxyatrazine ethylaminohydrolase(AtzB) (4) and N-isopropylammelide isopropylaminohydrolase (AtzC)(21). Atrazine chlorohydrolase, however, does not transformthe double dealkylated CAAT (23). Aminotriazines such as melamine(Fig. 1B) are hydrolyzed by melamine deaminase (TriA) from Acidovoraxavenae subsp. citrulli strain NRRL B-12227 (22). TriA has anamino acid sequence that is 98% identical to that of AtzA, yetit has a very different substrate specificity (22, 23).

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FIG. 1. Chemical structures of atrazine (A) and melamine (B).

In the present study, TriA, AtzB, and AtzC were found to hydrolyticallydisplace the one chloride and two amino substituents of CAAT.While all of these enzymes were previously observed to catalyzeonly deamination reactions, here we show that AtzB catalyzesa dechlorination reaction. The final product of these threereactions is cyanuric acid, which is readily mineralized bysoil bacteria.

Methods.

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Methods.

The substrate CAAT was obtained from Aldrich (Milwaukee, Wis.).2,4-Dichloro-6-amino-s-triazine (CCAT) was prepared from 2,4,6-trichloro-s-triazine(Aldrich) as previously described (26). CCAT was hydrolyzedto 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT) by adding a2.5 M equivalent of aqueous potassium hydroxide. The mixturewas chilled in an ice bath and acidified with acetic acid toproduce the precipitated CAOT. Ammelide was produced directlyfrom CCAT as described by Smolin and Rapoport (25). Ammelinewas prepared by acidifying CAAT in 20% (vol/vol) acetic acidin the presence of a potassium bromide catalyst, followed bybuffering with sodium acetate. Identity of the standard compoundswas confirmed by silination of the hydroxyl groups with 1,1,1,3,3,3-hexamethyldisilazane(Aldrich)/N,O-bis-trimethylsilyl-acetamide (Aldrich) and analysisof the resulting product by gas chromatography (GC)-mass spectrometry(MS). Synthetic standards and enzyme reaction products werealso analyzed by high-pressure liquid chromatography (HPLC)using a Hewlett-Packard 1100 Series system equipped with a photodiodearray detector interfaced to a Hewlett-Packard ChemStation.A Waters IC-PAK anion high capacity (HC) column (4.6 by 150mm) was used with a 100 mM phosphate buffer, pH 7, mobile phaseand a 1-ml min^-1 flow rate for detection of CAAT and CAOT. Thechlorinated triazines were detected at 255 and 200 nm, respectively.A 10 mM phosphate buffer, pH 5, mobile phase with a 0.25-mlmin^-1 flow rate was used to analyze ammeline and ammelide at200 nm. HPLC-MS was done in cooperation with Tom Krick at theMass Spectrometry Consortium for Life Sciences at the Universityof Minnesota. Ammonium acetate buffer was substituted as a mobilephase for MS detection. Ammonia release was detected as previouslydescribed (22). The triA gene was isolated as previously described(22), except that it was subsequently cloned into the EcoRI/HindIIIsites of a pTrc99A vector. The resulting vector, pJS5, was transformedinto Escherichia coli DH5 ${alpha}$ . Basal transcription levels producedoptimal soluble protein levels. Other recombinant strains usedin these studies were described elsewhere: AtzB (E. coli[pATZB2])(4) and AtzC (E. coli[pATZC]) (21) (N. Shapir, unpublished data).

Enzymatic reactions and formation of products were confirmedin both resting cell and cell extract experiments. Resting cellassays were conducted in 50 ml of 25 mM phosphate buffer, pH7, containing the given substrate. Cells were removed by centrifugationfor 2 min at 13,000 x g in a tabletop microcentrifuge, and thesupernatants were analyzed by HPLC and HPLC-MS. To prepare cellextracts, cells were harvested by centrifugation as describedabove, resuspended in ice-cold, 25 mM phosphate buffer, pH 7,and lysed by sonication (3- to 20-s bursts at 80% intensityon a Biosonik sonicator; Bronwill Scientific, Rochester, N.Y.).The resulting solution was centrifuged at 4°C to removecell debris (13,000 x g for 5 min in a tabletop microcentrifuge).The cell extract was incubated with substrate overnight andwas analyzed by HPLC and HPLC-MS. The identity of products wasconfirmed by comparison to standards. Bacteria for growth curvestudies were grown in R medium (24), with equimolar concentrationsof the various s-triazines as sole nitrogen sources. Sampleswere removed at specified time points and were analyzed spectophotometricallyat 600 nm. Due to the low water solubility of CAAT, maximumgrowth occurred at approximately an optical density at 600 nmof 0.15. Similar growth was observed with equimolar ammoniaconcentrations (data not shown). Substrate stability was testedover a period of 1 month. None of the compounds used throughoutthis study showed significant degradation in phosphate bufferat the pH values used over this time period. E. coli DH5 ${alpha}$ cells,lacking the recombinant genes, served as a control, and no degradationof the substrates was observed with this strain.

CAAT hydrolysis by TriA

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Caat hydrolysis by tria

. Resting cell suspensions of E. coli(pJS5) containing the triAgene were incubated with CAAT, and the culture filtrate wasanalyzed by HPLC and HPLC-MS. Over time, the CAAT peak disappearedwith the concomitant increase of another peak. The product wasidentified as CAOT by coelution with an authentic standard onHPLC, UV spectroscopy, and mass spectrometry. There was no furthermetabolism of CAOT by E. coli(pJS5). Additional experimentsshowed that ammonia release paralleled CAOT formation and thatE. coli(pJS5) grew on CAAT as a sole source of nitrogen forgrowth (Fig. 2).

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FIG. 2. Growth of recombinant E. coli on several potential nitrogen sources. Shown are results for E. coli(pJS5) (A) with CAAT as the sole nitrogen source, E. coli DH5 ${alpha}$ (B) with CAAT as the sole nitrogen source, E. coli(pATZB) (C) with CAOT as the sole nitrogen source, and E. coli(pATZC) (D) with ammelide as the sole nitrogen source (n = 3). Standard error bars are shown by vertical lines at each time point.

Grossenbacher et al. previously showed that the parent strainof triA, A. avenae subsp. citrulli strain NRRL B-12227 (formerlyPseudomonas sp. strain NRRLB-12227 and Pseudomonas sp. strainA) was capable of CAAT degradation, but the enzyme responsiblefor the activity was not identified (10). In the present study,we showed that TriA is the responsible enzyme. s-Triazine hydrolase(TrzA) from Rhodococcus corallinus NRRL B-15444R is a homologto TriA and also catalyzes turnover of CAAT (18). Data fromammonia release assays and UV spectrophotometry were used tohypothesize that the product of the TrzA reaction was CAOT;however, no standard was available in that study to confirmthe identity of the product. TrzA and TriA are homologous butshare only 45% sequence identity. TriA is much more highly relatedto AtzA, sharing 98% sequence identity. However, AtzA showsno detectable reactivity with CAAT (23).

It was previously reported that CAAT was degraded by TriA toyield ammelide (22). However, in the present work, we showedthat the acidic conditions used for the previous MS analysiscaused hydrolysis of the labile chloride substituent of CAOTto produce ammelide. The stability of CAOT at pH 7 was confirmedby experiments with standards, and MS at neutral pH identifiedthat CAOT was the only product produced by TriA. These observationsfurther support the conclusion that the distinct specificitiesof TriA, which catalyzes deamination reactions, and AtzA, whichcatalyzes dechlorination reactions, are determined by one ormore of the nine amino acids that differ between these two proteins.

CAOT hydrolysis by AtzB.

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Caot hydrolysis by atzb.

The enzymatic fate of CAOT has not been reported previously.In this study, CAOT was shown to be hydrolyzed by cell extractsprepared from an E. coli strain containing the enzyme AtzB.A parallel extract from a control E. coli strain lacking AtzBdid not hydrolyze CAOT. E. coli(pATZB2) transformed CAOT stoichiometricallyto ammelide as determined by UV spectroscopy and HPLC, withcomparison to standards. This was the only product detected.Consistent with the observation that only a chloride substituentwas removed, E. coli(pATZB2) did not grow on CAOT in the absenceof additional nitrogen sources (Fig. 2).

AtzB was previously identified for its ability to catalyze thedeamination of hydroxyatrazine to produce N-isopropylammelide(4). Results from these studies indicate that AtzB can hydrolyzeN-alkyl or chloride substituents and is less strict in its specificitythan AtzA or TriA. All three of these enzymes belong to thesame amidohydrolase superfamily, which includes such enzymesas adenosine deaminase and dihydroorotase (11, 21, 22). As thesuperfamily name suggests, most of these enzymes catalyze thehydrolysis of amino and amido groups. However, there is someprecedence for enzymes that physiologically catalyze deaminationreactions to also catalyze fortuitous dechlorination reactions(1, 2). It is remarkable in this respect that TriA shows a highdegree of specificity for deamination without any significantdechlorination activity.

Ammelide hydrolysis by AtzC.

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Ammelide hydrolysis by atzc.

Ammelide is known to be metabolized by bacteria, but the enzymesresponsible for its transformation have not been purified andcharacterized previously (7, 8). Here we show that purifiedAtzC from Pseudomonas sp. strain ADP catalyzes the hydrolyticdeamination of ammelide to produce cyanuric acid. Product identitywas confirmed with UV spectroscopy and HPLC, with comparisonto standards. The k_cat for the reaction was 2 s^-1, comparedto 14 s^-1 for the hydrolysis of N-isopropylammelide. Consistentwith these data, E. coli(pATZC) was observed to grow on ammelideas a sole nitrogen source (Fig. 2). A detailed purificationand characterization of AtzC will be published elsewhere.

Ammelide metabolism has been observed with numerous bacteria.The ammelide amidohydrolase gene (trzC) from A. avenae subsp.citrulli strain NRRL B-12227 has been cloned and sequenced (7)(GenBank protein accession number AAK00493), but the enzymewas not purified or characterized. It is shown in the presentstudy that AtzC has substantial activity with ammelide and willsupport bacterial growth on ammelide as the sole nitrogen source.The product of the AtzC reaction, cyanuric acid, is metabolizedby many soil bacteria. Genes and enzymes involved in the metabolismof cyanuric acid have been reported by several authors (5, 7,8, 12, 16).

Taken together, the data obtained in this study allow us todepict a complete hypothetical pathway for the metabolism ofCAAT (Fig. 3). This constellation of genes has not yet beendetected in any single bacterium. However, atzABC genes, knownto reside on a broad-host-range plasmid in Pseudomonas sp. strainADP (16), differ by only 9 nucleotides out of 4,081 from a hypotheticalclustering of triA, atzB, and atzC, rendering it plausible thatsuch a clustering exists in nature. Also TriA and TrzC are foundin the same bacterium, A. avenae subsp. citrulli strain NRRLB-12227. The addition of atzB to this bacterium would also resultin a functional pathway for CAAT.

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FIG. 3. Proposed hypothetical pathway for the degradation of CAAT.

A previous study showed that changes in a small number of aminoacids caused significant alterations in s-triazine substratespecificity for AtzA and TriA (20). The present study showsthat changes in the clustering of specific s-triazine-degradingenzymes can broaden the specificity of a metabolic pathway.The new pathway described here has potential environmental relevance,since CAAT is an important intermediate of s-triazine herbicidemetabolism in soil. Knowledge of enzyme sequence and substrateplasticity may increasingly be used to predict the course ofmetabolism in the environment.

ACKNOWLEDGMENTS

We thank Tom Krick of the Mass Spectrometry Consortium for LifeSciences at the University of Minnesota for his assistance inthe HPLC-MS analysis.

This research was supported in part by a grant from SyngentaCrop Protection, National Institutes of Health training grantGM08347, and a postdoctoral fellowship from the United States-IsraelBinational Agriculture Research and Development (BARD) fundgrant FI-295-99 (to N.S.).

FOOTNOTES

* Corresponding author. Mailing address: 140 Gortner Laboratory, 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.

REFERENCES

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