Applied and Environmental Microbiology, June 2003, p . 3653-3657, Vol . 69, No . 6

On the Origins of Cyanuric Acid Hydrolase: Purification, Substrates, and Prevalence of AtzD from Pseudomonas sp . Strain ADP

Isaac Fruchey,^1, Nir Shapir,^1,2,3 Michael J . Sadowsky,^1,3,4 and Lawrence P . Wackett^1,2,4*

Department of Biochemistry, Molecular Biology and Biophysics,² BioTechnology Institute,¹ Center for Microbial and Plant Genomics,⁴ Department of Soil, Water and Climate, University of Minnesota, St . Paul, Minnesota 55108³

Received 6 January 2003/ Accepted 6 March 2003

To understand the context of catabolic pathway evolution, it is necessary to delimit the substrate specificity of the pathway enzymes . Most enzymes turn over more than one substrate . Examining the suite of substrates for all pathway enzymes defines the set that can traverse the entire catabolic pathway and, thus, provide physiological benefit to the host organism . In this context, it is also necessary to know if any metabolites are natural products and thus might have served as bacterial growth substrates during millions of years of evolutionary history . It is also important to know something about the distribution of the relevant enzyme(s) in different bacterial strains . This study investigates AtzD, one of the two bacterial enzymes currently known to hydrolyze cyanuric acid, an intermediate formed during microbial metabolism of melamine and s-triazine herbicides .

s-Triazine herbicides such as atrazine are anthropogenic chemicals without any known natural product source . The complete catabolic pathway for atrazine degradation in Pseudomonas sp . strain ADP is known; all the genes have been sequenced; and two of the enzymes have been purified, and their substrate specificities have been determined (32, 38) . There is evidence that AtzA, a dehalogenase that initiates metabolism of atrazine, evolved recently and is highly specific for s-triazine herbicide substrates (29) . AtzC, or N-isopropylammelide amidohydrolase, catalyzes the third reaction in the atrazine catabolic pathway (24) . Eight substrates were found to be reactive with AtzC, and cyanuric acid was a product with each of them (32) .

The metabolism of cyanuric acid to carbon dioxide and ammonia by Pseudomonas sp . strain ADP has been shown to proceed via three consecutive amide bond-cleaving reactions catalyzed by AtzD, AtzE, and AtzF in crude extracts of recombinant Escherichia coli (17, 18) . AtzD from Pseudomonas sp . strain ADP (accession no . AAK50331) appears to be isofunctional with TrzD (accession no . AAC61577) from Pseudomonas sp . strain NRRLB-12227 . The latter has been purified from a recombinant E . coli strain containing the trzD gene and shown to hydrolyze cyanuric acid to biuret (14) . AtzD shares 56% sequence identity with TrzD . Thus, while these enzymes clearly share homology and functionality, they are likely separated by millions of years of evolution . The only other proteins in the GenBank database with obvious sequence relatedness to AtzD and TrzD are barbiturase from Rhodococcus erythropolis (accession no . CAC86669) (34), an AtzD homolog in the Bradyrhizobium japonicum genome (accession no . NP_77392), and a TrzD homolog from a Chelatobacter sp . (accession no . AAK52819) (24), with sequence identities ranging from 40 to 60% in comparison to AtzD from Pseudomonas strain ADP . Barbiturase catalyzes the hydrolysis of barbituric acid, an intermediate in the oxidative catabolism of pyrimidines (8, 35) . Barbituric acid structurally resembles cyanuric acid (Fig . 1) and is a tight-binding inhibitor of TrzD, with a K_i of 0.1 µM .

 
There are a large number of cyclic amides which undergo enzyme-catalyzed hydrolytic ring-opening reactions (Fig . 1) . The intermediates and reactions depicted in Fig . 1 are widespread in the biological world . For example, all organisms which have been investigated biochemically contain pyrimidine rings, such as those acted on by hydantoinase (5,6-dihydropyrimidinase) . E . coli contains at least three enzymes known to hydrolyze cyclic amides, although for some the physiologically relevant substrate is unclear (15) . In the present study, we considered three questions relevant to the evolutionary history of AtzD . (i) Does AtzD catalyze ring-opening reactions with, or is it inhibited by, other cyclic amides? (ii) Do other cyclic amidases hydrolyze cyanuric acid? (iii) Do a range of phylogenetically unrelated bacteria that grow on cyanuric acid contain genes homologous to atzD or trzD?

To answer the first question, we cloned and overexpressed the atzD gene from Pseudomonas sp . strain ADP in E . coli, purified the enzyme to homogeneity, performed general characterization of the isolated enzyme, and examined its reactivity with different cyclic amides . The atzD gene was amplified by PCR using the forward and reverse primers 5'-CATGTATCACATCGACG-3' and 5'-ACAGAGACCGAATTCCT-3', respectively . The 1.1-kb fragment was directly ligated into the pGEM T Easy PCR cloning vector (Promega, Madison, Wis.) . The gene was digested with EcoRI and cloned into the same site in the expression vector pKK223-3 (Amersham Pharmacia, Piscataway, N.J.) to generate pIF1 . When E . coli JM109(pIF1) was grown at 37°C on Luria-Bertani agar plates (26) containing isopropyl-ß-D-thiogalactopyranoside (IPTG) and 17.5 mg of cyanuric acid per ml, a clearing zone was seen around the colonies after 3 days . E . coli cells not containing the atzD gene did not show clearing . For enzyme purification studies, the cells were grown in Luria-Bertani broth containing 2 mM IPTG to yield approximately 7% of the soluble protein as AtzD .

AtzD was purified 14-fold from a crude soluble extract of E . coli JM109(pIF1) with a total recovery of 7% (Table 1) . A single band corresponding to a molecular weight of 44,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Each enzyme subunit contained less than 0.1 atom of any transition metal, Mg, or Ca as determined by inductively coupled plasma emission spectroscopy . The metal chelators EDTA and o-phenanthroline did not diminish activity . Furthermore, no stimulation of enzyme activity was observed upon addition of zinc(II), copper(II), iron(II), cobalt(II) or nickel(II) salts . Steady-state kinetic assays (31) with AtzD were conducted at optimum buffer and pH conditions, 25 mM Tris buffer at pH 8.2, as determined in preliminary experiments . The k_cat and K_m values for cyanuric acid were found to be 6.8 s^-1 ± 0.7 s^-1 and 57 ± 10 µM, respectively . The product of the reaction was shown to be biuret by comparing its properties to those of an authentic standard by using UV spectroscopy, high-pressure liquid chromatography, and thin-layer chromatography .

 
We next tested the reactivity of AtzD with a series of compounds structurally analogous to cyanuric acid, some of which are natural-product cyclic amides . Commercial compounds tested were of the highest purity available from Aldrich Chemical Company (Milwaukee, Wis.) . N-Methyl-, N,N-dimethyl- and N,N,N-trimethylcyanuric acid were synthesized as previously described (33) . The compounds were tested as substrates by measuring a decrease in the initial absorbance of 1.0 with a Beckman (Fullerton, Calif.) DU640 UV-visible spectrophotometer set at the maximum absorption of each compound in the UV region (14) . Assays were conducted in 25 mM Tris buffer, pH 8.2 . Positive results obtained by spectrophotometry were confirmed by monitoring the disappearance of the substrate by high-pressure liquid chromatography with a Hewlett-Packard (San Fernando, Calif.) HP 1100 system equipped with a photodiode array detector interfaced to an HP ChemStation . Compounds were tested as potential inhibitors by coincubation with cyanuric acid either at time zero or after preincubation of the potential inhibitor with AtzD prior to the addition of cyanuric acid . Steady-state kinetic parameters were determined by using standard initial-rate data treatment methods (31) . Purified AtzD was found to be unreactive with the following cyclic amide and triazine compounds as substrates: cytosine, 5-azacytosine, thymine, uracil, 5,6-dihydroxyuracil, 2,4,5-trihydroxypyrimidine, 6-azauracil, ammeline, ammelide, 2,4-dioxohexahydro-s-triazine, trimethoxycyanuric acid, trithiocyanuric acid, barbituric acid, 5-nitrobarbituric acid, hydantoin, succinamide, maleimide, and parabanic acid . Karns (14) previously tested TrzD with uracil, ammeline, and ammelide and found no evidence of reactivity . Barbituric acid had been observed to be a tight-binding competitive inhibitior of TrzD (14) . It was observed in the present study to also be a strong competitive inhibitor of AtzD with a K_i of 0.2 µM . Moreover, maleimide was found to be a time-dependent, mixed-type inhibitor of AtzD .

N-Methylisocyanuric acid was found to be an excellent substrate for AtzD, with a k_cat of 3.1 s^-1 ± 0.3 s^-1 and a K_m of 71 ± 10 µM . The k_cat/K_m of 4.4 x 10⁴ s^-1 M^-1 with N-methylisocyanuric acid was of the same order of magnitude as that measured with cyanuric acid . However, N,N-dimethyl- and N,N,N-trimethyl isocyanuric acid showed no discernible reactivity with AtzD, indicating a high degree of substrate discrimination by this enzyme .

To answer the second in our series of questions, experiments were subsequently conducted to determine if several other known cyclic amidases showed activity with cyanuric acid used as the substrate (Fig . 1) . Purified hydantoinase (recombinant, immobilized from E . coli; Fluka, Milwaukee, Wis.), allantoinase (from E . coli; provided by Robert Hausinger, Michigan State University), ß-lactamase (type II from Bacillus subtilis; Sigma, St . Louis, Mo.), and creatinase (from Flavobacterium species; Sigma) showed no detectable activity with cyanuric acid . Previously, barbiturase (EC 3.5.2.1) was shown not to have activity with cyanuric acid as its substrate despite the close structural similarity of barbituric acid and cyanuric acid (Fig . 1) and the moderately high sequence identity of 41% for barbiturase and AtzD .

Collectively, the data described above, when added to previously published studies (14, 21, 23), are consistent with the idea that AtzD and TrzD are the two major enzyme homologs found in bacterial populations that hydrolyze cyanuric acid . Since AtzD and TrzD are 44% different in amino acid sequence, their evolutionary divergence is not recent . The prevalence of AtzD and TrzD in bacterial populations able to metabolize cyanuric acid was tested here by using specific PCR primers that would selectively amplify the atzD or trzD genes . The primers used to amplify atzD were, for AtzD392f, 5'-ACGCTCAGATAACGGAGA-3' and, for AtzD949r, 5'-TGTCGGAGTCACTTAGCA-3' . The size of the amplified fragment was 558 bp . The primers used to amplify trzD were, for TrzD274f, 5'-CACTGCACCATCTTCACC-3' and, for TrzD936r, 5'-GTTACGAAC CTCACCGTC-3' . The size of the amplified fragment was 663 bp . Despite the taxonomic and geographic diversity of the bacteria tested, all the strains contained either an atzD or trzD gene homolog, but not both (Table 2) . These data strongly suggest that the atzD and trzD genes are geographically widespread and prevalent in phylogenetically distinct bacteria metabolizing cyanuric acid . Rousseaux and coworkers (23) used PCR methods and hybridization to show that DNA similar to the trzD gene was present in gram-negative bacterial strains isolated from French soils that had the ability to grow on atrazine . Ostrofsky and coworkers (21), by using a trzD gene probe, concluded that this gene alone could not universally account for all the cyanuric acid degradation phenotypes in their soils, consistent with our observations here .

 
In the present paper, we show that AtzD from Pseudomonas sp . strain ADP has very strict substrate specificity . That observation, coupled with our previous report that the atzD, -E, and -F genes are contiguous on plasmid pADP-1 and may be coordinately controlled (17), is consistent with the idea that AtzD, AtzE, and AtzF function primarily to metabolize cyanuric acid to 3 mol of carbon dioxide and 3 mol of ammonia, the latter supporting growth as a nitrogen source . Cyanuric acid is reported to be a natural product (39) . Although its origin in soils is unknown, purines and pyrimidines are commonly found in soil (27), and uric acid is known to undergo oxidation by hydrogen peroxide and potassium permanganate to yield cyanuric acid (12) . Regardless of its origin, cyanuric acid was found in all soils tested at levels of at least 6.5 ppm (39) . Moreover, the study by Wise and Walters was conducted in 1917—before s-triazine herbicides were applied in the environment, which began around 1960 . Since that time, billions of pounds of s-triazine herbicides have been manufactured and applied to soils . Additionally, cyanuric acid is generated in significant quantities from the decomposition of dichloro- and trichloroisocyanuric acids, which are used as chlorinating agents in swimming pools (4, 11) . There has also been some input of synthetic cyanuric acid into soil as a slow-release fertilizer (1) . In this context, microbial cyanuric acid metabolism might be anticipated to be widespread . In fact, numerous laboratory studies have demonstrated the growth of bacteria and fungi with cyanuric acid as the sole nitrogen source (5, 6, 7, 9, 10, 13, 16, 19, 20, 22, 23, 25, 36, 40, 41, 42; M . L . de Souza, N . R . Pechacek, L . P . Wackett, M . J . Sadowsky, and B . L . Hoyle, Abstr . 98th Gen . Meet . Am . Soc . Microbiol., abstr . Q-195, p . 453, 1998) .

The present study provides insights into the origins and underlying molecular basis of bacterial cyanuric acid metabolism . While AtzD is active with N-methylisocyanuric acid, it is not active with any other cyclic amides that are known to be generated in bacteria via intermediary metabolic reactions, such as barbituric acid . In fact, the cyclic amides maleimide and barbituric acid are potent inhibitors of AtzD . Moreover, the atzD gene is clustered with atzE and -F and appears to be coordinately regulated with those genes (17) . These studies suggest that AtzD is not recently evolved like AtzA (30) and that microbial enzymes active with cyanuric acid may have existed for a long period of time . It is perhaps relevant to these observations that recent experiments to mimic the chemical conditions of ancient Earth have generated cyclic amides such as cyanuric acid (37) .

We thank Robert Hausinger for providing purified E . coli allantoinase and Gil Johnson for synthesis of the methylcyanuric acids . We thank Jennifer Seffernick, Mark Radosevich, Kyria Boundy-Mills, David Crowley, Richard Eaton, and Thomas Moorman for bacterial strains used in this study .

Present address: Battelle Memorial Institute, Aberdeen, MD 21001 .

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