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Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine. doi:10.1128/JB.01243-09

Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The [k.sub.cat] and [K.sub.m] of AtzB with hydroxyatrazine as a substrate were 3 [s.sup.-1] and 20 [micro]M, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.

TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 [micro]M zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a [k.sub.cat]/[K.sub.m] of 1.1 x [10.sup.4] [s.sup.-1] [M.sup.-1], and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced [k.sub.cat]/ [K.sub.m] of 21 [s.sup.-1] [M.sup.-1]. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, [Mn.sup.2+] and [Fe.sup.2+], respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate super-family); each monomer has four [beta][alpha][beta][beta][beta] modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H[214.sup.NE1] and E[267.sup.OE1]; one axial ligand, H[155.sup.NE1]; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 [Angstrom]), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An 'open' coordination site trans to E267 is the likely binding site for [O.sup.2]. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound [O.sup.2] during catalysis are identified and discussed in the context of current mechanistic hypotheses.

Results reveal that N-isopropylammelide aminohydrolase reacts with a variety of s-triazine substrates cleaving the side chain carbon-nitrogen bonds. It also reacts with s-triazine pesticides. The enzyme contains a five-coordinate metal center and the activity is dependent on the zinc moiety.

Research has been conducted on the catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. The complete sequencing and annotation of this plasmid have been carried out in understanding the assembly of the atrazine catabolic pathway and the results of the sequence analysis are reported.

Research demonstrates that melamine deaminase and atrazine chlorohydrolase of Pseudomonas sp. strain NRRL B-12227 and strain ADP, respectively, show 9 amino acid residue difference between them and catalyze totally different substrate compounds. Data suggest that they are two identical proteins but catalyze distinct biochemical reactions, deamination and dehalogenation, respectively.

Atrazine is a herbicide that has the potential to contaminate soil and water. Degradation of this pollutant is achieved in Pseudomonas sp via a stepwise process culminating in the production of cyanuric acid. Three genes have been shown to code for the enzymes involved in atrazine biodegradation. These are designated as atzA, -B and -C. Genetic surveys were conducted to determine if other organisms possess atrazine degrading characteristics. Results reveal that these genes are ubiquitous and highly conserved in nature.

The third gene (atzC) in the atrazine catabolic pathway was cloned from a Pseudomonas sp strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The findings indicate that AtzA, AtzB and AtzC diverged from a single ancestor. Moreover, these enzymes underwent reconstitution onto an atrazine catabolic plasmid by random events for the purpose of metabolizing the herbicide atrazine.

The toluene dioxygenase-catalyzed oxidation of various halogenated ethenes, propenes, butenes and nonhalogenated cis-2-pentene, an isomeric mix of 2-hexenes, cis-2-heptene and cis-2-octene were evaluated as substrates for toluene dioxygenase. Substrates lacking a halogen substituent on sp2 carbon atoms were dioxygenated, while those with halogen and one or more unsubstituted allylic methyl groups were monooxygenated to produce allylic alcohols.

The reaction of trichloroethylene (TCE) with purified toulene 2-monooxygenase components from Burkholderia cepacia was analyzed to determine the kinetic parameters and products of TCE oxidation. Analysis of the steady-state kinetics of TCE oxidation involving purified toluene 2-monooxygenase and reduced nicotinamide adenine dinucleotide indicated the formation of carbon monoxide, formate and glyoxylate. Furthermore, TCE oxidation involved the TCE-dependent inactivation of toluene 2-monooxygenase and the generation of TCE epoxide and formyl chloride.

Purified atrazine chlorohydrolase from Pseudomonas sp. strain ADP is 1,419 nucleotides and encodes a 473-amino-acid protein and catalyzes the dechlorination of atrazine to hydroxyatrazine through hydrolysis. This hydrolytic dechlorination is useful for metabolizing atrazine, leading to environmental restoration. Atrazine, deethylatrazine, deisopropylatrazine, and simazine are hydrolyzed by purified atrazine chlorohydrolase.

Sequence analysis of the manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase from Arthrobacter globiformis and a study of the relationship between the extradiol dioxygenases that are iron- and manganese-dependent reveals that the manganese-dependent enzyme from A. globiformis is a member of the major extradiol dioxygenase family. Sequencing of fragments of two genes on either side of the (3,4-DHPA) 2,3-dioxygenase-encoding mndD gene, the mndE and mndC genes, reveals 46 and 37% amino acid sequence similarity with the XylG and XylJ of Pseudomonas putida TOL plasmid. TOL plasmid genes exhibit an evolutionary association with Arthrobacter genes.

Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 µM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 µM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. [ABSTRACT FROM AUTHOR]

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class ofmetallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment. [ABSTRACT FROM AUTHOR]

The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by (13)C nuclear magnetic resonance ((13)C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria.