scholarly journals Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Asim K Bera ◽  
Kelly G. Aukema ◽  
Mikael Elias ◽  
Lawrence P. Wackett
Keyword(s):  
Cyanuric Acid ◽  
Exit Channel ◽  
Acid Hydrolase

scholarly journals Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp. Strain Mel

2011 ◽  
Vol 78 (5) ◽  
pp. 1397-1403 ◽  
Author(s):  
Anthony G. Dodge ◽  
Lawrence P. Wackett ◽  
Michael J. Sadowsky
Keyword(s):  
454 Pyrosequencing ◽  
Minimal Medium ◽  
Doubling Time ◽  
Cyanuric Acid ◽  
Acid Hydrolase ◽  
Content Type ◽  
N Source ◽  
Genes Encoding ◽  
Rhodococcus Sp ◽  
Roche 454

ABSTRACTRhodococcussp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase genetrzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ∼265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. TheRhodococcussp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.

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

2012 ◽  
Vol 194 (17) ◽  
pp. 4579-4588 ◽  
Author(s):  
J. L. Seffernick ◽  
J. S. Erickson ◽  
S. M. Cameron ◽  
S. Cho ◽  
A. G. Dodge ◽  
...  
Keyword(s):  
Sequence Space ◽  
Cyanuric Acid ◽  
Protein Family ◽  
Acid Hydrolase ◽  
Reaction Products ◽  
Defining Sequence

scholarly journals Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Adi Radian ◽  
Kelly G. Aukema ◽  
Alptekin Aksan ◽  
Lawrence P. Wackett
Keyword(s):  
Escherichia Coli ◽  
Cyanuric Acid ◽  
Uv Spectroscopy ◽  
Water Disinfection ◽  
Enzyme Inactivation ◽  
Amine Group ◽  
Acid Hydrolase ◽  
Whole Cell ◽  
Content Type ◽  
Degradation Rates

ABSTRACTChlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treatedEscherichia colicells expressing cyanuric acid hydrolase (CAH) fromMoorella thermoaceticaexhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulatedE. colicells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications.IMPORTANCEHypochlorite is used in vast quantities for water disinfection, killing bacteria on surfaces, and washing and whitening. In pools, spas, and other waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance.

scholarly journals Cyanuric Acid Biodegradation via Biuret: Physiology, Taxonomy, and Geospatial Distribution

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Kelly G. Aukema ◽  
Lambros J. Tassoulas ◽  
Serina L. Robinson ◽  
Jessica F. Konopatski ◽  
Madison D. Bygd ◽  
...  
Keyword(s):  
Cyanuric Acid ◽  
Current Knowledge ◽  
Model Organism ◽  
The United States ◽  
Degradation Pathway ◽  
Acid Hydrolase ◽  
Single Model ◽  
Acid Degradation ◽  
Degrading Bacteria

ABSTRACT Cyanuric acid is an industrial chemical produced during the biodegradation of s-triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a single model system, Pseudomonas sp. strain ADP, in which cyanuric acid hydrolase (AtzD) opens the s-triazine ring and AtzEG deaminates the ring-opened product. A significant question remains as to whether the metabolic pathway found in Pseudomonas sp. ADP is the exception or the rule in bacterial genomes globally. Here, we show that most bacteria utilize a different pathway, metabolizing cyanuric acid via biuret. The new pathway was determined by reconstituting the pathway in vitro with purified enzymes and by mining more than 250,000 genomes and metagenomes. We isolated soil bacteria that grow on cyanuric acid as a sole nitrogen source and showed that the genome from a Herbaspirillum strain had a canonical cyanuric acid hydrolase gene but different flanking genes. The flanking gene trtB encoded an enzyme that we show catalyzed the decarboxylation of the cyanuric acid hydrolase product, carboxybiuret. The reaction generated biuret, a pathway intermediate further transformed by biuret hydrolase (BiuH). The prevalence of the newly defined pathway was determined by cooccurrence analysis of cyanuric acid hydrolase genes and flanking genes. Here, we show the biuret pathway was more than 1 order of magnitude more prevalent than the original Pseudomonas sp. ADP pathway. Mining a database of over 40,000 bacterial isolates with precise geospatial metadata showed that bacteria with concurrent cyanuric acid and biuret hydrolase genes were distributed throughout the United States. IMPORTANCE Cyanuric acid is produced naturally as a contaminant in urea fertilizer, and it is used as a chlorine stabilizer in swimming pools. Cyanuric acid-degrading bacteria are used commercially in removing cyanuric acid from pool water when it exceeds desired levels. The total volume of cyanuric acid produced annually exceeds 200 million kilograms, most of which enters the natural environment. In this context, it is important to have a global understanding of cyanuric acid biodegradation by microbial communities in natural and engineered systems. Current knowledge of cyanuric acid metabolism largely derives from studies on the enzymes from a single model organism, Pseudomonas sp. ADP. In this study, we obtained and studied new microbes and discovered a previously unknown cyanuric acid degradation pathway. The new pathway identified here was found to be much more prevalent than the pathway previously established for Pseudomonas sp. ADP. In addition, the types of environment, taxonomic prevalences, and geospatial distributions of the different cyanuric acid degradation pathways are described here.

scholarly journals Bacterial Cyanuric Acid Hydrolase for Water Treatment

2015 ◽  
Vol 81 (19) ◽  
pp. 6660-6668 ◽  
Author(s):  
Sujin Yeom ◽  
Baris R. Mutlu ◽  
Alptekin Aksan ◽  
Lawrence P. Wackett
Keyword(s):  
Cyanuric Acid ◽  
Porous Silica ◽  
Silica Matrix ◽  
Water Disinfection ◽  
Significant Activity ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Content Type ◽  
Silica Bead ◽  
Degradation Rates

ABSTRACTDi- 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 fromAcidovorax citrullistrain 12227, AtzD fromPseudomonassp. strain ADP, and CAH fromMoorella thermoaceticaATCC 39073. Each enzyme was expressed recombinantly inEscherichia coliand 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 thermophileM. thermoaceticaretained 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.

scholarly journals Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

2013 ◽  
Vol 195 (23) ◽  
pp. 5233-5241 ◽  
Author(s):  
A. G. Dodge ◽  
C. S. Preiner ◽  
L. P. Wackett
Keyword(s):  
Cyanuric Acid ◽  
Protein Family ◽  
Acid Hydrolase ◽  
Fungal Kingdom

scholarly journals Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073

2009 ◽  
Vol 75 (22) ◽  
pp. 6986-6991 ◽  
Author(s):  
Qingyan Li ◽  
Jennifer L. Seffernick ◽  
Michael J. Sadowsky ◽  
Lawrence P. Wackett
Keyword(s):  
Low Temperatures ◽  
Cyanuric Acid ◽  
Water Remediation ◽  
Acid Hydrolase ◽  
Swimming Pools ◽  
Acid Hydrolases ◽  
Metabolic Intermediate ◽  
Moorella Thermoacetica ◽  
Trichloroisocyanuric Acid ◽  
Structurally Stable

ABSTRACT 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.

scholarly journals Cyanuric Acid Hydrolase from Azorhizobium caulinodans ORS 571: Crystal Structure and Insights into a New Class of Ser-Lys Dyad Proteins

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e99349 ◽  
Author(s):  
Seunghee Cho ◽  
Ke Shi ◽  
Jennifer L. Seffernick ◽  
Anthony G. Dodge ◽  
Lawrence P. Wackett ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cyanuric Acid ◽  
Acid Hydrolase ◽  
Azorhizobium Caulinodans ◽  
New Class

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

2016 ◽  
Vol 82 (6) ◽  
pp. 1638-1645 ◽  
Author(s):  
Jennifer L. Seffernick ◽  
Lawrence P. Wackett
Keyword(s):  
Cyanuric Acid ◽  
The United States ◽  
Microbial Populations ◽  
Initial Reaction ◽  
Protein Fold ◽  
Acid Hydrolase ◽  
Content Type ◽  
Pyrimidine Catabolism ◽  
Filtration System ◽  
Bacteria And Fungi

ABSTRACTCyanuric 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 thes-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 others-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 fromMoorella thermoaceticais being tested for this purpose.

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