scholarly journals High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Thomas S. Peat ◽  
Sahil Balotra ◽  
Matthew Wilding ◽  
Carol J. Hartley ◽  
Janet Newman ◽  
...  
Keyword(s):  
Barbituric Acid ◽  
Cyanuric Acid ◽  
Acid Hydrolase ◽  
Phylogenetic Distribution ◽  
Class V ◽  
Content Type ◽  
X Ray ◽  
Cyclic Amide ◽  
Amide Hydrolase ◽  
Hydrolase Family

ABSTRACTThe Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from aRhodococcusspecies and a class V cyclic amide hydrolase (2.4 Å) from aFrankiaspecies for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. TheFrankiacyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.IMPORTANCEThe Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.

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 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 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 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.

scholarly journals X-Ray Structure of the Amidase Domain of AtzF, the Allophanate Hydrolase from the Cyanuric Acid-Mineralizing Multienzyme Complex

2014 ◽  
Vol 81 (2) ◽  
pp. 470-480 ◽  
Author(s):  
Sahil Balotra ◽  
Janet Newman ◽  
Nathan P. Cowieson ◽  
Nigel G. French ◽  
Peter M. Campbell ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Cyanuric Acid ◽  
Kluyveromyces Lactis ◽  
Multienzyme Complex ◽  
Content Type ◽  
X Ray ◽  
Terminal Domain ◽  
Catalytic Function ◽  
Dead End

ABSTRACTThe activity of the allophanate hydrolase fromPseudomonassp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization ofs-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase ofKluyveromyces lactis, when purified enzyme was testedin vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.

scholarly journals Effects of thermal treatments on characteristics and morphological variations in the deposits of urea-SCR systems

2021 ◽  
Author(s):  
Sadashiva Prabhu S ◽  
Kapilan Natesan ◽  
Nagaraj Shivappa Nayak
Keyword(s):  
Cyanuric Acid ◽  
Catalytic Reduction ◽  
Morphological Changes ◽  
Water Solution ◽  
Slight Variation ◽  
Gravimetric Analysis ◽  
Oxides Of Nitrogen ◽  
Deposit Formation ◽  
X Ray ◽  
Exhaust Gases

AbstractSelective catalytic reduction (SCR) systems are employed by automobile manufacturers for the abatement of environmental pollutants like oxides of nitrogen (NOx) emitted from exhaust gases of diesel engines. In SCR, the urea-water solution (UWS) is injected to exhaust gases in the form of a spray to generate the reducing agent NH3. Deposit formation at lower temperatures is a major concern with this technology. The deposits not only create backpressure but also leak NH3 to the environment as they deplete. It is very important to know the depletion characteristics of deposits formed at lower temperatures in order to assess the NH3 leakage to the environment when the engine exhaust gases attain higher temperatures. In the present work, deposits formed at a low-temperature range of 150–200°C for continuous run along with UWS injection were investigated. Additionally, they were aged at 300°C in the absence of UWS to check the variation in characteristics with the rise of temperature. By gravimetric analysis, it is inferred that the deposits formed at higher pre-age temperatures are less prone to depletion as the temperature increases. The elemental analysis using energy-dispersive X-ray spectroscopy (EDX) indicates slight variation in carbon, nitrogen and oxygen compositions for all the pre-age conditions. As an extended study, the byproducts at pre-age and post-age conditions were investigated through X-ray diffraction (XRD). The compounds like cyanuric acid (CYA) and biuret were not observed when pre-age samples were aged at 300°C. Instead, the compounds like ammelide, ammeline, triuret and melamine were observed. Scanning electron microscope (SEM) study revealed morphological changes in both pre-age and post-age samples. Further, the crystallinity variations were also observed for the changes in the heating cycles during deposit formation. The gravimetric analysis of deposits in pre-age and post-age conditions helps in predicting the amount of deposits for transient load cycles.

Characteristic properties of AlTiN and TiN coated HSS materials

2015 ◽  
Vol 67 (2) ◽  
pp. 172-180 ◽  
Author(s):  
Mumin Sahin ◽  
Cenk Misirli ◽  
Dervis Özkan
Keyword(s):  
Surface Roughness ◽  
Tensile Strength ◽  
High Speed ◽  
Cutting Tools ◽  
High Speed Steel ◽  
Wear Properties ◽  
Content Type ◽  
X Ray ◽  
Number Of Cycles ◽  
Future Work

Purpose – The purpose of this paper is to examine mechanical and metallurgical properties of AlTiN- and TiN-coates high-speed steel (HSS) materials in detail. Design/methodology/approach – In this study, HSS steel parts have been processed through machining and have been coated with AlTiN and TiN on physical vapour deposition workbench at approximately 6,500°C for 4 hours. Tensile strength, fatigue strength, hardness tests for AlTiN- and TiN-coated HSS samples have been performed; moreover, energy dispersive X-ray spectroscopy and X-ray diffraction analysis and microstructure analysis have been made by scanning electron microscopy. The obtained results have been compared with uncoated HSS components. Findings – It was found that tensile strength of TiAlN- and TiN-coated HSS parts is higher than that of uncoated HSS parts. Highest tensile strength has been obtained from TiN-coated HSS parts. Number of cycles for failure of TiAlN- and TiN-coated HSS parts is higher than that for HSS parts. Particularly TiN-coated HSS parts have the most valuable fatigue results. However, surface roughness of fatigue samples may cause notch effect. For this reason, surface roughness of coated HSS parts is compared with that of uncoated ones. While the average surface roughness (Ra) of the uncoated samples was in the range of 0.40 μm, that of the AlTiN- and TiN-coated samples was in the range of 0.60 and 0.80 μm, respectively. Research limitations/implications – It would be interesting to search different coatings for cutting tools. It could be the good idea for future work to concentrate on wear properties of tool materials. Practical implications – The detailed mechanical and metallurgical results can be used to assess the AlTiN and TiN coating applications in HSS materials. Originality/value – This paper provides information on mechanical and metallurgical behaviour of AlTiN- and TiN-coated HSS materials and offers practical help for researchers and scientists working in the coating area.

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

Study in performance and morphology of polyamide 12 produced by selective laser sintering technology

2018 ◽  
Vol 24 (5) ◽  
pp. 813-820 ◽  
Author(s):  
Junjie Wu ◽  
Xiang Xu ◽  
Zhihao Zhao ◽  
Minjie Wang ◽  
Jie Zhang
Keyword(s):  
Selective Laser Sintering ◽  
Polarized Light ◽  
Chain Scission ◽  
High Energy ◽  
Laser Sintering ◽  
X Ray Diffraction ◽  
Scanning Calorimetry ◽  
Content Type ◽  
X Ray ◽  
Polyamide 12

Purpose The purpose of this paper is to investigate the effect of selective laser sintering (SLS) method on morphology and performance of polyamide 12. Design/methodology/approach Crystallization behavior is critical to the properties of semi-crystalline polymers. The crystallization condition of SLS process is much different from others. The morphology of polyamide 12 produced by SLS technology was investigated using scanning electron microscopy, polarized light microscopy, differential scanning calorimetry, X-ray diffraction and wide-angle X-ray diffraction. Findings Too low fill laser power brought about bad fusion of powders, while too high energy input resulted in bad performance due to chain scission of macromolecules. There were three types of crystal in the raw powder material, denoted as overgrowth crystal, ring-banded spherulite and normal spherulite. Originality/value In this work, SLS samples with different sintering parameters, as well as compression molding sample for the purpose of comparison, were made to study the morphology and crystal structure of sintered PA12 in detail.

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