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MINIREVIEW

Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism

N. Shapir,^1,2 E. F. Mongodin,³ M. J. Sadowsky,^1,2 S. C. Daugherty,³ K. E. Nelson,^3, and L. P. Wackett^1,2*

The BioTechnology Institute,¹ Center for Microbial and Plant Genomics, University of Minnesota, St. Paul, Minnesota 55108,² The Institute for Genomic Research, Rockville, Maryland³

	INTRODUCTION

Top Introduction References

 
In recent years, studies on the catabolism of organonitrogen compounds have accelerated due to the recognition that these compounds are widespread and significant environmentally. Nitrogen is the second most abundant macronutrient in cellular organisms, where it is found in major macromolecules (proteins and nucleic acids), primary metabolites (ATP and NADH), and secondary metabolites, such as antibiotics. Well over 50,000 organonitrogen compounds are biosynthesized by plants and microbes (27). Many of these compounds are N-heterocycles, ring structures containing one or more nitrogen atoms in the rings. A significant fraction of anthropogenic compounds, commodity chemicals and pharmaceuticals, are N-heterocycles. One class of industrial N-heterocycles, also known as s-triazines, contain the 1,3,5-triazine ring.

Humans introduced s-triazine herbicides, such as atrazine (Fig. 1), into the environment one-half century ago. Commercial s-triazines characteristically contain ring carbon atoms that contain substituents other than hydrogen (Fig. 1, upper left). This fundamental ring structure resembles the 1,3-diazenes, or pyrimidines, and as such, their metabolic transformation by microbes should be reasonably facile. However, s-triazine compounds were initially found to be poorly biodegradable. In the decades following its introduction, measured environmental half-lives of atrazine were variable and relatively long, typically 60 to 400 days (28, 35, 41, 46, 67, 69). Soil metabolite and biochemical studies provided evidence for nonspecific oxidation reactions leading to partial metabolism. More recently, environmental half-lives have decreased dramatically; they are now typically measured at 1 to 50 days (4, 32, 48, 80). Additionally, metabolite profiles have changed. In the period between 1960 and 1990, many studies reported bacterial metabolism of atrazine to occur via dealkylation of the N-alkyl substituents on the s-triazine ring (20, 21, 34), a pathway that does not typically lead to s-triazine ring cleavage (44, 62). Since 1995, most reports of metabolic pathways for atrazine degradation have described metabolism via hydroxyatrazine and not involving dealkylation (18, 49, 53, 56, 70, 72, 73, 75, 76). Earlier reports expressed the view that isolating bacteria able to grow on atrazine as a sole nitrogen or carbon source was difficult (13, 20). Most recently, pure cultures of bacteria capable of growing on atrazine could be obtained from most soils (39). These observations are consistent with the idea that a new metabolic pathway for atrazine catabolism may have evolved and spread in recent evolutionary times.

View larger version (16K): [in this window] [in a new window]   FIG. 1. General structure of s-triazine compounds undergoing microbial metabolism (upper left, inset) and the most broadly demonstrated pathway for catabolism of the herbicide atrazine.

	s-TRIAZINE METABOLISM AND GENES

 
Most commonly, the bacterial metabolism of commercial s-triazine compounds is reported to occur via enzymatic, hydrolytic displacement of substituents from the three carbon atoms of the s-triazine ring (Fig. 1). The reactions occur in an obligate order. For example, metabolism of the herbicide atrazine is initiated by a hydrolytic dechlorination reaction to produce hydroxyatrazine (Fig. 1). The environmental detection of hydroxyatrazine has thus become diagnostic for this metabolic pathway (36). The broad-specificity enzymes TrzN, AtzB, and AtzC can funnel dozens of s-triazine ring compounds into cyanuric acid, where they are acted on by a set of enzymes with much narrower substrate specificities. The enzyme AtzD was shown to have activity only with cyanuric acid and N-methylisocyanuric acid (22), and the homologous, isofunctional enzyme TrzD was not active with any substrate tested other than cyanuric acid (33). AtzD acts in concert with AtzE and AtzF to hydrolyze cyanuric acid to yield 3 moles each of carbon dioxide and ammonia (40). Cyanuric acid was previously thought to be hydrolyzed to urea, but more recent studies have shown that all bacteria studied deaminate biuret to generate allophanic acid as an intermediate (9). Allophanic acid is the substrate for AtzF or TrzF and is enzymatically hydrolyzed to carbon dioxide and ammonia (63) (Fig. 1).

There are differences in organization and regulation between the genes encoding the broad-specificity enzymes (Fig. 1, right) and narrow-specificity enzymes (Fig. 1, left). The narrow-specificity enzymes are encoded by the atzDEF genes, which are contiguous on the plasmid pADP-1 and have been shown to be coregulated under the control of the upstream atzR gene product (23, 40). Expression of the atzDEF genes is controlled by nitrogen limitation and the presence of cyanuric acid as an inducer (23). The broad-specificity genes show very different behavior. First, the genes were observed not to be contiguous on plasmid pADP-1, immediately suggesting lack of an operonic structure for the upper-pathway genes. Moreover, experiments using Northern blot hybridization and reverse transcriptase PCR indicate that the atzA, atzB, and atzC genes are expressed constitutively (19, 40). These observations are consistent with the view that AtzA, AtzB, and AtzC are enzymes that have evolved their specific substrate preferences in recent times, perhaps since s-triazine herbicides were introduced into the environment about 50 years ago. While AtzB and AtzC displace a range of alkylamino groups from s-triazine rings, they are not reactive with the corresponding pyrimidine substrates. It has also been observed that the dehalogenase AtzA is closely related to TriA, an enzyme that catalyzes deamination reactions (58). It was further shown in the laboratory that two amino acid changes are sufficient to evolve TriA, which is not appreciably active in dehalogenation, into an enzyme with significant dehalogenase activity (51). These two amino acid changes are present in the naturally occurring AtzA enzymes that have been sequenced to date (61). In general, the data suggest that the AtzA, AtzB, and AtzC enzymes function in bacteria specifically for s-triazine metabolism and that their genes have not had sufficient time to physically accrete into a highly organized, operonic-type structure.

The atzA, atzB, and atzC genes were observed to be flanked by insertion sequence elements in plasmid pADP-1 in Pseudomonas sp. strain ADP. Identical insertion sequences have been found flanking the atz genes in other bacteria (76, 79), suggesting that mobile gene cassettes have transferred between strains. The linkage of the trzN-atzB-atzC genes on a plasmid element in Arthrobacter aurescens TC1 was established by bacterial artificial chromosome cloning and sequencing (54). This has been corroborated by the complete genome sequencing of A. aurescens TC1 (43), which has also provided the opportunity to analyze s-triazine metabolism in the context of the entire metabolic network of a single bacterium. It had not been previously possible to do this, as discussed in more detail below.

	ENORMOUS METABOLIC VERSATILITY FROM THREE CATABOLIC GENES AND COMPLEMENTARY CORE METABOLISM

 
Most bacteria have been isolated on atrazine and related s-triazines as the sole N source but will not grow on these compounds as the sole carbon source (2, 13, 16, 37, 50, 73, 76). A. aurescens TC1 is exceptional, utilizing various s-triazines as both sole carbon and nitrogen sources. A. aurescens TC1 was isolated at a roadside spill site, where 1,000 pounds of atrazine had contaminated 35 cubic yards of soil; the site was bioremediated to acceptable levels with approval by the U.S. Environmental Protection Agency (71, 72). The method of treatment in that study was a recombinant Escherichia coli strain containing highly expressed AtzA. Prior to addition to the soil, the recombinant bacterium was treated with the cross-linking agent glutaraldehyde, which served to both kill the cells and stabilize atrazine chlorohydrolase activity.

A. aurescens TC1 was reported, in the initial study describing its isolation, to grow on 22 different s-triazine compounds. A. aurescens TC1 degrades atrazine most extensively of all microorganisms described to date; a growing culture degraded 3 g atrazine per liter over the course of 1 week (72). What underlies this efficient s-triazine metabolism by A. aurescens TC1? The trzN, atzB, and atzC genes, encoding atrazine catabolism to cyanuric acid (Fig. 1), are localized on the large plasmid pTC1. Sequencing the complete plasmid by both shotgun techniques and the sequencing of bacterial artificial chromosome clones spanning certain regions established that six copies of the trzN gene are present on plasmid pTC1, and this might lead to higher expression levels of TrzN (43). TrzN has recently been purified in active, recombinant form and shown to require zinc for activity. When TrzN is fully populated with zinc, it has a k_cat of 25 s^–1 (65), which is an order of magnitude higher than that of AtzA, which has a k_cat of 2 s^–1 (18). The combined gene dosage and k_cat enhancement may provide an increase of almost 2 orders of magnitude in atrazine dehalogenation by A. aurescens TC1. This perhaps reflects the fact that A. aurescens TC1 was isolated from soil contaminated with 1,000 pounds of atrazine for more than 1 year prior to its isolation.

A. aurescens TC1 accumulates cyanuric acid stoichiometrically, and thus, when grown solely on atrazine, must derive all of its carbon, nitrogen, and energy from the N-alkyl fragments liberated by AtzB and AtzC (Fig. 2, top). It is remarkable that this metabolism is so broad: as deduced from genomic annotation and bench experiments, A. aurescens TC1 is estimated to grow on over 500 distinct s-triazine compounds (Fig. 2, bottom). This derives from a combination of (i) the broad substrate specificity of TrzN, AtzB, and AtzC and (ii) a large array of enzymes known to metabolize a diverse set of primary amines. To start this metabolism, the first enzyme must react with a wide range of substrates. TrzN was first investigated by Topp and coworkers (75) and was shown to have a significantly broader substrate range than AtzA. A more recent study expanded the known substrate range of TrzN to include other herbicides and analogs of commercial herbicides (66). At least eight distinct leaving groups are hydrolytically displaced from carbon 2 of the s-triazine ring, and TrzN tolerates a wide range of different N-alkyl groups at the 4 and 6 carbon atoms.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Metabolism encoded by plasmid genes trzN, atzB, and atzC showing the number of substituent permutations known to be processed by each enzyme. TrzN will displace the eight leaving groups shown and tolerate most combinations of the N-alkyl substituents displaced by AtzB and AtzC, except when R1 = R2 = H or R1 = R2 = CH3.

When TrzN, AtzB, and AtzC are combined in the same cell, each with its own broad substrate tolerances, the number of theoretically acceptable permutations is 560, as illustrated in Fig. 2. This number is based on what is currently known about the substituents tolerated by TrzN and takes into account N-alkyl substituents known to be hydrolytically removed by AtzB and AtzC. The number is considered theoretical because not all of the 560 s-triazine compound permutations have been tested. However, 64 different s-triazine ring compounds, more than 11% of the theoretically likely substrates, have been synthesized and shown to be substrates for the different enzymes. There is no evidence of inhibition by a substrate of one enzyme on another. The s-triazine ring hydrolases are thought to be expressed constitutively (40). All of the liberated amines support growth, as reported previously (72). The overall breadth of amine metabolism by A. aurescens TC1 is discussed below.

Many of the recently isolated atrazine-degrading bacteria contain TrzN rather than AtzA for initiating the metabolism of s-triazines (1, 56, 70, 72). In one recent example with simazine as the herbicide and a number of different bacteria, TrzN, but not AtzA, was detected in the strains (55). Previously, AtzA, the atrazine chlorohydrolase from Pseudomonas sp. strain ADP, was purified, characterized, and found in diverse bacterial genera prior to the discovery of TrzN (17, 18). The substrate range of AtzA is quite restricted; only chloro and fluoro substituents were found to be displaced (59). Thus, bacteria expressing AtzABC would be unable to metabolize many nonchlorinated s-triazine herbicides, for example, ametryn, prometryn, and atratone. These herbicides, along with atrazine, can be catabolized by cells containing TrzN-AtzB-AtzC. The observations of increasing prevalence of TrzN in isolates is consistent with, but does not prove, the idea that TrzN may be becoming more widespread environmentally because of superior catalytic performance.

TrzN and AtzA are both members of the amidohydrolase superfamily. However, the amino acid sequence relatedness between them is only 27%, so they have apparently evolved independently from different nearest ancestors (65). AtzA shows an astounding 98% amino acid sequence identity to melamine deaminase, an enzyme that was not observed to catalyze appreciable dechlorination (61). The natural evolution of a deaminase to a dechlorinase was recapitulated by laboratory directed-evolution experiments that identified the key amino acid residues changing the activity into a dechlorinase (51). A similarly close relative of TrzN has not been identified. TrzN requires one equivalent of zinc per subunit for maximal activity. AtzA contains a single atom of ferrous iron per subunit. This is consistent with their assignment to the amidohydrolase superfamily, where almost all proteins are known to contain a mononuclear or binuclear metal center at the respective active sites (68). A significant number of amidohydrolase family members catalyze the hydrolytic removal of an amino group from nitrogen heterocyclic rings. This might underlie nature's choice of this enzyme superfamily for the evolution of an initiating reaction to deal with s-triazine herbicides. AtzB and AtzC also fall into the amidohydrolase superfamily. These reactions, releasing amines, are more in keeping with reactions catalyzed by related enzymes in the amidohydrolase superfamily.

The potential of the enzymatic triad of TrzN-AtzB-AtzC to promote the survival of A. aurescens TC1, which does not metabolize cyanuric acid, is directly linked to their ability to liberate assimilatable amines (Fig. 2). The s-triazine herbicides generally provide n-alkyl, branched-alkyl, and hydroxyalkyl amines. Other s-triazines have been synthesized for purposes of investigating the properties of the enzymes. The metabolisms of diverse amines are discussed below.

	AMINE METABOLISM BY ARTHROBACTER AURESCENS TC1 AND OTHER BACTERIA

 
A. aurescens TC1 can grow on atrazine as the sole source of nitrogen and carbon, unlike Pseudomonas sp. strain ADP, which requires supplemental carbon with atrazine. A. aurescens TC1 could grow on many other s-triazine herbicides, such as simazine, terbuthylazine, propazine, cyanazine, ametryn, prometryn, and terbutryn (72). All of the common s-triazine herbicides are secondary amines. Biodegradation of the herbicides liberates primary alkylamines via the broad-specificity enzymes AtzB and AtzC (Fig. 2). It was observed that A. aurescens TC1 grows on primary alkylamines more rapidly than s-triazines, indicating that amines are oxidized more rapidly than they are eliminated from s-triazine rings (72). In contrast, the metabolism of atrazine by Pseudomonas sp. strain ADP leads to transient accumulation of isopropylamine (16). This may partly explain why the fixation of s-triazine catabolism genes in A. aurescens TC1 has led to highly efficient metabolism.

Genomic sequencing and growth experiments with A. aurescens TC1 revealed unprecedented amine metabolism capabilities. Interestingly, a significant number of bacteria isolated for growth on alkyl or aromatic amines and polyamines have been Arthrobacter strains (15, 24, 26, 31, 38, 45, 82). Other high-G+C gram-positive bacteria, Pseudomonas spp., and Klebsiella spp., have also been identified for their metabolism of amines (14, 25).

Several enzyme classes have been shown to be involved in amine metabolism; for example, copper amine oxidases (6), flavoprotein dehydrogenases (57), and the pathway mediated by -glutamyl-isopropylamide synthetase (14). The first two enzyme classes had previously been demonstrated in Arthrobacter sp. The genome sequencing of A. aurescens TC1 revealed the presence of all three classes of amine-metabolizing enzymes. The most metabolically complex is the Ipu pathway initiated by -glutamyl-isopropylamide synthetase (Fig. 3, top). This pathway involves amine amidation via reaction with L-glutamate, alkyl group oxidation, deamidation, and amine liberation. The cluster of genes encoding these related enzymes are designated as isopropyl amine utilization (ipu) genes because the metabolic activity was first elucidated with isopropylamine. This unique combination of genes, encoding an amide synthetase and a multicomponent cytochrome P450 monooxygenase system, plus alcohol and aldehyde dehydrogenases, makes this cluster relatively easy to identify during genome annotation. Despite this, and the sequencing and annotation of hundreds of microbial genomes, this gene cluster is very rare. The ipu-dependent pathway has previously been described in only one bacterium, Pseudomonas sp. strain KIE171 (14). Remarkably, A. aurescens TC1 contains two Ipu pathway gene clusters. Each cluster is localized to one of the two plasmids, pTC1 or pTC2 (Fig. 3).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Genes, enzymes, and pathways in A. aurescens TC1 proposed to be involved in metabolism of amines liberated from s-triazine compounds by AtzB and AtzC. The gene designations refer to GenBank loci in accession numbers CP000474 (chromosome and plasmid), CP000475 (pTC1), and CP000476 (plasmid pTC2.)

Both copper amine oxidases and the flavoprotein amine dehydrogenases catalyze the oxidation of an amine to the corresponding carbonyl compound with the liberation of a less-substituted amine or to ammonia from primary amines (Fig. 3, bottom). A. aurescens TC1 contains several amine flavoprotein dehydrogenases that are probably involved in the metabolism of dimethylglycine, sarcosine, and choline, based on genome annotation and precedents in other Arthrobacter species reported in the literature (42, 52).

At least five amine oxidases have been identified in A. aurescens TC1, and they are more likely to be involved in the metabolism of primary alkylamines of the type liberated during the metabolism of s-triazine herbicides (Fig. 3). Arthrobacter amine oxidases, denoted by annotation as methylamine, histamine, phenethylamine, and tyramine oxidases, have had prototypes purified and characterized (12, 15, 78, 81). The names of these enzymes were derived from the substrates they were first tested with, but their substrate specificities overlap. For example, methylamine oxidases that oxidize methyl, ethyl, propyl, and butylamine have been described (15).

The X-ray structures of several Arthrobacter copper amine oxidases have been determined (6). They all share similar active-site architectures with a copper metal and a topoquinone cofactor. The topoquinone cofactor serves to form an enzyme-bound imine, followed by liberation of the carbonyl product and ammonia. Because of the similarities in structures and mechanism among the different copper amine oxidases, it is difficult to discern which of the five amine oxidases that have been identified are most active with the many different amines liberated during s-triazine metabolism. Substrate specificity is determined, in this class, by the length of the channel leading into the active-site topoquinone cofactor, and it is difficult to predict this structural feature from sequence information alone.

In general, it is not possible to predict the relative involvement of all the different amine-metabolizing enzyme systems in the catabolism of specific amines. It is likely that many of them are involved in, and contribute to, a broad-based amine metabolism. In this context, experimental studies have substantiated the breadth of amine metabolism predicted in A. aurescens TC1 by genome annotation (72; K. Borchert and N. Shapir, unpublished data). A. aurescens TC1 was observed to grow on the following alkylamines: ethyl-, propyl-, isopropyl-, butyl-, and hexylamines. The bacterium grew on the following di- and polyamines: putrescine, cadaverine, and spermine. A. aurescens TC1 grew on benzylamine, 2-phenethylamine, and histamine. This broad metabolic capability is consistent with the assignments made by genome annotation.

	ORGANONITROGEN METABOLISM BY ARTHROBACTER SP. EXTENDED TO OTHER PESTICIDES

 
The deposited genome sequence of Arthrobacter sp. strain FB24 (GenBank accession no. NZ_AAHG00000000) reveals multiple amine oxidases and dehydrogenases in that strain. Another Arthrobacter genomic effort provided the complete sequence of the 160-kb nicotine (NIC) plasmid of Arthrobacter nicotinovorans (30). The NIC plasmid harbors genes for oxidation of the nitrogen heterocyclic ring of nicotine and the amine fragments thus generated (5, 10, 11, 30); it encodes amine dehydrogenases of the type found in A. aurescens TC1. The data suggest that amine oxidation is a very important metabolic niche for Arthrobacter spp. in soil.

Figure 4 (the central circle) illustrates that the metabolic capabilities of Arthrobacter for organonitrogen functional groups extends beyond amines. There are extensive capabilities to metabolize amides, carbamates, ureides, -amino acids, and ß-amino acids. The central circle of Fig. 4 represents a metabolic "core" that many Arthrobacter strains may possess. In A. aurescens TC1, the genes encoding these core reactions are largely chromosomal genes. Overlaid on this core, extending the catabolic-substrate range of the organism, A. aurescens TC1 possesses plasmid genes predicted to encode the uptake and metabolism of amino acids, cyclic guanidines, arylamines, nitriles, and substituted s-triazine ring compounds (Fig. 4, top). The intracellular metabolites from of that transport or catabolism are amino acids, guanidines, amines, amides, and ureas that are catabolized by enzymes encoded by genes found in the chromosome of A. aurescens TC1 and Arthrobacter FB24.

View larger version (49K): [in this window] [in a new window]   FIG. 4. Features of nitrogen assimilation by Arthrobacter showing plasmid-encoded metabolism (outside the circle) and chromosome-encoded metabolism (inside the circle). The lower half outside the circle represents metabolisms from different Arthrobacter strains, where the metabolism was established to be plasmid encoded (11-15). The upper half outside the circle represents metabolism from A. aurescens TC1, predicted by annotation to be encoded by plasmid genes.

	CONCLUSIONS

 
s-Triazines are anthropogenic chemicals that are now known to be readily recycled by many soil bacteria. The genes and enzymes underlying this metabolism have been elucidated. There is evidence that s-triazine metabolism has recently evolved, and the initiating reactions are almost invariably plasmid encoded. Arthrobacter species are being increasingly isolated and are particularly efficient at metabolizing s-triazine compounds (1, 8, 53, 72). This efficiency stems from the broad-spectrum TrzN enzyme initiating metabolism and from the ability to rapidly assimilate the alkylamine fragments generated by AtzB and AtzC. The recent genome sequences of Arthrobacter strains help to reveal the core metabolism that underlies efficient s-triazine metabolism. The nitrogen-metabolic genes on the Arthrobacter chromosome also reveal why this genus of bacteria has consistently been isolated for the catabolism of a wide range of organonitrogen chemicals found in the environment.

	ACKNOWLEDGMENTS

 
We acknowledge Gil Johnson for the synthesis of s-triazine compounds and Karen Borchert for help in growth experiments.

This work was supported by grant 0333161 from the National Science Foundation and a grant from Syngenta Crop Protection.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-1700. E-mail: wacke003{at}umn.edu.

Published ahead of print on 17 November 2006.

Present address: Department of Biology, Howard University, Washington, DC.

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