Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO(2) and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K(m) of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4, 6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204?kDa and 160?kDa. Each was a tetramer or pentamer of 44?kDa and 33?kDa, respectively.     

Characterization of a novel melamine-degrading bacterium isolated from a melamine-manufacturing factory in China

Melamine (2,4,6-triamino-1,3,5-triazine, C₃H₆N₆), belonging to the s-triazine family, is an anthropogenic and versatile raw material for a large number of consumer products and its extensive use has resulted in the contamination of melamine in the environment. A novel melamine-degrading bacterium strain CY1 was isolated from a melamine-manufacturing factory in China. The strain is phylogenetically different from the known melamine-degrading bacteria. Approximately, 94 % melamine (initial melamine concentration 4.0 mM, initial cell OD 0.05) was degraded in 10 days without the addition of additional carbon source. High-performance liquid chromatography showed the production of degradation intermediates including ammeline, ammelide, cyanuric acid, biuret, and urea. Kinetic simulation analysis indicated that transformation of urea into ammonia was the rate-limiting step for the degradation process. The melamine–cyanurate complex was formed due to self-assembly of melamine and cyanuric acid during the degradation. The tracking experiment using CY1 cells and ¹³C₃-melamine showed that the CY1 could mineralize s-triazine ring carbon to CO₂. The strain CY1 could also catalyze partial transformation of cyromazine, a cyclopropyl derivative of melamine, to 6-(cyclopropylamino)-[1,3,5]triazine-2,4-diol.     

Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

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 ¹³C nuclear magnetic resonance (¹³C-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.     

Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α₂ and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.     

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

Cyanuric acid hydrolase (AtzD) from Pseudomonas sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.     

Mineralization of s-triazine herbicides by a newly isolated Nocardioides species strain DN36

A novel s-triazine-mineralizing bacterium—Nocardioides sp. strain DN36—was isolated from paddy field soil treated with ring-U-¹⁴C-labeled simetryn ([¹⁴C]simetryn) in a model paddy ecosystem (microcosm). In a tenfold-diluted R2A medium, strain DN36 liberated ¹⁴CO₂ from not only [¹⁴C]simetryn but also three ring-U-¹⁴C-labeled s-triazines: atrazine, simazine, and propazine. We found that DN36 mineralized ring-U-¹⁴C–cyanuric acid added as an initial substrate, indicating that the bacterium mineralized s-triazine herbicides via a common metabolite, namely, cyanuric acid. Strain DN36 harbored a set of genes encoding previously reported s-triazine-degrading enzymes (TrzN-AtzB-AtzC), and it also transformed ametryn, prometryn, dimethametryn, atraton, simeton, and prometon. The findings suggest that strain DN36 can mineralize a diverse range of s-triazine herbicides. To our knowledge, strain DN36 is the first Nocardioides strain that can individually mineralize s-triazine herbicides via the ring cleavage of cyanuric acid. Further, DN36 could not grow on cyanuric acid, and the degradation seemed to occur cometabolically.     

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines

Cyanuric acid was likely present on prebiotic Earth, may have been a component of early genetic materials, and is synthesized industrially today on a scale of more than one hundred million pounds per year in the United States. In light of this, it is not surprising that some bacteria and fungi have a metabolic pathway that sequentially hydrolyzes cyanuric acid and its metabolites to release the nitrogen atoms as ammonia to support growth. The initial reaction that opens the s-triazine ring is catalyzed by the unusual enzyme cyanuric acid hydrolase. This enzyme is in a rare protein family that consists of only cyanuric acid hydrolase (CAH) and barbiturase, with barbiturase participating in pyrimidine catabolism by some actinobacterial species. The X-ray structures of two cyanuric acid hydrolase proteins show that this family has a unique protein fold. Phylogenetic, bioinformatic, enzymological, and genetic studies are consistent with the idea that CAH has an ancient protein fold that was rare in microbial populations but is currently becoming more widespread in microbial populations in the wake of anthropogenic synthesis of cyanuric acid and other s-triazine compounds that are metabolized via a cyanuric acid intermediate. The need for the removal of cyanuric acid from swimming pools and spas, where it is used as a disinfectant stabilizer, can potentially be met using an enzyme filtration system. A stable thermophilic cyanuric acid hydrolase from Moorella thermoacetica is being tested for this purpose.     

Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.     

Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073

Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications.s-Triazine compounds have diverse applications as herbicides, resins, and disinfectants. The s-triazine herbicides, such as atrazine, help to promote high-yield, sustainable agriculture. Melamine, or triamino-s-triazine, is a high-volume industrial chemical. Melamine-based polymers have outstanding thermosetting properties, ideal for their use in kitchen utensils and plates, as high-pressure laminates such as Formica, and as whiteboards. Di- and tri-chloroisocyanuric acids find widespread application as disinfectants, algicides, and bactericides. The chlorinated isocyanuric acids are used in wastewater treatment, in the textile industry as bleaching compounds, and in preventing and curing diseases in husbandry and fisheries. A major use of these compounds is for swimming pool chlorination. They have outstanding performance for maintaining a high, stable chlorine content by dissolving slowly in water, allowing a continuous metered dosing of chlorine.Degradation of these and other s-triazine compounds results in the production of cyanuric acid (Fig. ​(Fig.1).1). Cyanuric acid has come under increased scrutiny because of its potential involvement in comediating toxicity resulting from the ingestion of melamine (10). Recently, melamine has been found in adulterated pet food and baby formula. Melamine and its metabolite cyanuric acid cocrystallize at low concentrations and are implicated in acute renal failure in cats that have consumed adulterated food products (10). Cyanuric acid degradation is also of interest from the perspective of environmental remediation. The use of di- or trichloroisocyanuric acid in pool water results in spontaneous chemical dechlorination that disinfects the water but also produces, as a by-product, large amounts of cyanuric acid. High levels of cyanuric acid perturb the equilibrium, thus preventing dechlorination by additional chlorinated isocyanuric acid, such that disinfection is not achieved. As a result, swimming pools must be emptied and refilled, using water and causing discharge issues. It would be desirable to remediate pool water in situ, conserving water, saving money, and extending pool water use. In this context, there is a need to better understand cyanuric acid degradation and to identify highly stable biological catalysts to use for this purpose.Open in a separate windowFIG. 1.Atrazine, ametryn, trichloroisocyanuric acid, and melamine are all metabolized via cyanuric acid that is transformed to biuret by the action of cyanuric acid hydrolases.Microbial enzymatic degradation of cyanuric acid has been studied previously (3, 4, 8, 18). Two distinct but homologous enzymes, AtzD from Pseudomonas sp. strain ADP (8) and TrzD from Pseudomonas sp. strain NRRLB-12227 (now called Acidovorax avenae subsp. citrulli) (11), have been studied in detail. These enzymes, known as cyanuric acid hydrolases, catalyze the conversion of cyanuric acid to biuret (Fig. ​(Fig.1).1). Biuret is not considered toxic to humans and degrades more readily than cyanuric acid.Barbiturase is the only protein known to be homologous to cyanuric acid hydrolase that has a defined and different physiological function. Barbiturase catalyzes the conversion of barbituric acid to ureidomalonic acid in organisms that catabolize pyrimidines by the oxidative pathway. Barbiturase is unstable at 4°C in the absence of ethylene glycol and dithiothreitol (DTT). Furthermore, activity is completely lost when the protein is maintained at 55°C for 30 min (20). AtzD and TrzD are relatively stable at 4°C, but they lose activity when frozen (our unpublished data). Moreover, the thermostability properties of AtzD and TrzD are not well studied, but these enzymes are derived from mesophilic bacteria. In this context, we initiated a search to identify a stable cyanuric acid hydrolase. Enzymes that are more stable in response to temperature changes are more stable in response to many environmental factors. Thus, a thermostable enzyme would be most applicable to pool water and other remediation efforts.We employed bioinformatic techniques that identified a cyanuric acid hydrolase homolog in Moorella thermoacetica ATCC 39073, an anaerobic, acetogenic bacterium that is able to grow at 65°C. The gene was cloned into E. coli, the protein was expressed at high levels, the recombinant E. coli strain degraded cyanuric acid, and the enzyme was obtained in homogeneous form by a convenient one-step purification. The enzyme''s function as a cyanuric acid hydrolase was confirmed, and it was shown to be significantly more stable than other known members of the cyanuric acid protein family.     

Purification and Characterization of an Inducible s-Triazine Hydrolase from Rhodococcus corallinus NRRL B-15444R

The widespread use and relative persistence of s-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted s-triazines have been identified. Rhodococcus corallinus NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-N-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related s-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.     

Isolation and Characterization of Atrazine Mineralizing Bacillus subtilis Strain HB-6

Atrazine is a widely used herbicide with great environmental concern due to its high potential to contaminate soil and waters. An atrazine-degrading bacterial strain HB-6 was isolated from industrial wastewater and the 16S rRNA gene sequencing identified HB-6 as a Bacillus subtilis. PCR assays indicated that HB-6 contained atrazine-degrading genes trzN, atzB and atzC. The strain HB-6 was capable of utilizing atrazine and cyanuric acid as a sole nitrogen source for growth and even cleaved the s-triazine ring and mineralized atrazine. The strain demonstrated a very high efficiency of atrazine biodegradation with a broad optimum pH and temperature ranges and could be enhanced by cooperating with other bacteria, suggesting its huge potential for remediation of atrazine-contaminated sites. To our knowledge, there are few Bacillus subtilis strains reported that can mineralize atrazine, therefore, the present work might provide some new insights on atrazine remediation.     

A novel amidohydrolase (DmhA) from Sphingomonas sp. that can hydrolyze the organophosphorus pesticide dimethoate to dimethoate carboxylic acid and methylamine

Objectives

To characterize a novel dimethoate amidohydrolase from Sphingomonas sp. DC-6.

Results

A gene, dmhA, encoding the dimethoate amidohydrolase responsible for transforming dimethoate to dimethoate carboxylic acid and methylamine, was cloned from Sphingomonas sp. DC-6. Sequence analysis and molecular modeling indicate that DmhA shares 31–57 % amino acid sequence identities with other functionally confirmed amidohydrolase. DmhA was expressed in Escherichia coli BL21 (DE3) and purified by Ni–NTA affinity chromatography. The purified DmhA could hydrolyze 4-acetaminophenol, dimethoate and propanil. DmhA activity was optimal at 30 °C and pH 7.5. Hg²⁺, Zn²⁺, Cu²⁺, Cd²⁺, Tween 80, Triton X-100 or SDS strongly inhibited its activity. The K _m and k _cat values of DmhA for dimethoate are 0.02 mM and 1.2 s⁻¹, respectively.

Conclusions

DmhA was confirmed to be a novel dimethoate amidohydrolase which could eliminate the toxicity of dimethoate, providing a novel gene resource for the development of pesticide-degrading enzyme preparation and mechanistic study of dimethoate hydrolysis.

     

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

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 (13)C 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 of metallohydrolases. 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.     

Biochemical and Genetic Characterization of trans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain

trans-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The K_m and kcat values of this enzyme for trans-2′-carboxybenzalpyruvate were 50 μM and 13 s⁻¹, respectively. trans-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2′-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.     

Nitrite Elimination and Hydrolytic Ring Cleavage in 2,4,6-Trinitrophenol (Picric Acid) Degradation

Two hydrogenation reactions in the initial steps of degradation of 2,4,6-trinitrophenol produce the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. The npdH gene (contained in the npd gene cluster of the 2,4,6-trinitrophenol-degrading strain Rhodococcus opacus HL PM-1) was shown here to encode a tautomerase, catalyzing a proton shift between the aci-nitro and the nitro forms of the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. An enzyme (which eliminated nitrite from the aci-nitro form but not the nitro form of the dihydride complex of 2,4,6-trinitrophenol) was purified from the 2,4,6-trinitrophenol-degrading strain Nocardioides simplex FJ2-1A. The product of nitrite release was the hydride Meisenheimer complex of 2,4-dinitrophenol, which was hydrogenated to the dihydride Meisenheimer complex of 2,4-dinitrophenol by the hydride transferase I and the NADPH-dependent F₄₂₀ reductase from strain HL PM-1. At pH 7.5, the dihydride complex of 2,4-dinitrophenol is protonated to 2,4-dinitrocyclohexanone. A hydrolase was purified from strain FJ2-1A and shown to cleave 2,4-dinitrocyclohexanone hydrolytically to 4,6-dinitrohexanoate.     

Isolation of a novel gene encoding a 3,5,6-trichloro-2-pyridinol degrading enzyme from a cow rumen metagenomic library

3,5,6-trichloro-2-pyridinol (TCP) is a major metabolite of the insecticide chlorpyrifos and is hazardous to human and animal health. A gene encoding a TCP degrading enzyme was cloned from a metagenomic library prepared from cow rumen. The gene (tcp3A) is 2.5 kb in length, encoding a protein (Tcp3A) of 599 amino acid residues. Tcp3A has a potential signal sequence, as well as a putative ATP/GTP binding site, and a likely amidation site. The molecular weight of the enzyme is 62 kDa by SDS–PAGE. Comparison of Tcp3A with the NCBI database using BLASTP revealed homology to amidohydrolase proteins. Recombinant Escherichia coli harboring the tcp3A gene could utilize TCP as the sole source of carbon. TLC and HPLC revealed that TCP was degraded by recombinant E. coli harboring tcp3A. This is the first report of a gene encoding a TCP degrading enzyme.