The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Lawrence P. Wackett - One of the best experts on this subject based on the ideXlab platform.
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Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel
Scientific Reports, 2017Co-Authors: Asim K. Bera, Kelly G. Aukema, Mikael Elias, Lawrence P. WackettAbstract:Cyanuric Acid Hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric Acid, a stabilizer for the chlorine. Degradation of excess cyanuric Acid is necessary to maintain chlorine disinfection in the waters. Cyanuric Acid Hydrolase opens the cyanuric Acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric Acid Hydrolase from Acidovorax citrulli. The crystal structure at 2.19 A resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric Acid ring, and the mobile lysine guides products through the exit channel.
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Bacterial Cyanuric Acid Hydrolase for Water Treatment
Applied and Environmental Microbiology, 2015Co-Authors: Sujin Yeom, Baris R. Mutlu, Alptekin Aksan, Lawrence P. WackettAbstract:ABSTRACT 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.
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Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom
Journal of Bacteriology, 2013Co-Authors: Anthony G. Dodge, Chelsea S. Preiner, Lawrence P. WackettAbstract: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.
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defining sequence space and reaction products within the cyanuric Acid Hydrolase atzd barbiturase protein family
Journal of Bacteriology, 2012Co-Authors: Jennifer L. Seffernick, Michael J. Sadowsky, Anthony G. Dodge, Jasmine S Erickson, Stephan M Cameron, Jack E Richman, Lawrence P. WackettAbstract: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 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.
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Thermostable cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073
Applied and Environmental Microbiology, 2009Co-Authors: Qingyan Li, Jennifer L. Seffernick, Michael J. Sadowsky, Lawrence P. WackettAbstract: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.
Juan S Bonifacino - One of the best experts on this subject based on the ideXlab platform.
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requirement of the human garp complex for mannose 6 phosphate receptor dependent sorting of cathepsin d to lysosomes
Molecular Biology of the Cell, 2008Co-Authors: Javier F Perezvictoria, Gonzalo A Mardones, Juan S BonifacinoAbstract:The biosynthetic sorting of Acid Hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the Acid Hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.
Bruce W. Trotman - One of the best experts on this subject based on the ideXlab platform.
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Pigment gallstone disease.
Gastroenterology clinics of North America, 1991Co-Authors: Bruce W. TrotmanAbstract:Black and brown pigment gallstones are morphologically, compositionally, and clinically distinct. Black stones form primarily in the gallbladder in sterile bile and are associated with advanced age, chronic hemolysis, alcoholism, cirrhosis, pancreatitis, and total parenteral nutrition. Brown stones form not only within the gallbladder but also within the intrahepatic and extrahepatic ducts; they are uniformly infected with enteric bacteria and are usually associated with ascending cholangitis. Brown stones are related to juxtapapillary duodenal diverticula and are the predominant type of de novo common bile duct stones. Cholecystectomy is usually curative in black pigment stone disease, whereas stones often recur after cholecystectomy for brown stone disease. The pathogenesis of black stones is probably related to nonbacterial, nonenzymatic hydrolysis of bilirubin conjugates. At the pH of bile, this results in two monohydrogenated bilirubin anions that precipitate with calcium ions. Bilirubin monoconjugates that are increased in several conditions, such as Gilbert's syndrome and chronic hemolysis, may play a pivotal role in black stone formation as a source of unconjugated monohydrogenated bilirubin and as a possible co-precipitant with calcium. The precipitation of calcium carbonate and phosphate is influenced by local gallbladder factors. Brown pigment stones are formed in bile infected with enteric bacteria that elaborate hydrolytic enzymes: beta-glucuronidase, phospholipase A, and conjugated bile Acid Hydrolase. The resulting anions of bilirubin and fatty Acids form insoluble calcium salts. We used nb/nb mice with a chronic hemolytic anemia as a model of hemolysis-induced black stone disease. The presence of 40% bilirubin monoconjugates in mouse gallstones indicated the importance of this moiety in the pathogenesis of black stones. Other data obtained by marrow transplantation experiments in mice revealed the relative importance of genotype versus the hemolytic anemia on determinants such as biliary bile Acid composition and mucin secretory glands in the mouse gallbladder neck. Additional physical chemical studies of the interaction of unconjugated bilirubin in model bile solutions will be helpful in further delineating the pathogenesis of both black and brown pigment gallstones.
Javier F Perezvictoria - One of the best experts on this subject based on the ideXlab platform.
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requirement of the human garp complex for mannose 6 phosphate receptor dependent sorting of cathepsin d to lysosomes
Molecular Biology of the Cell, 2008Co-Authors: Javier F Perezvictoria, Gonzalo A Mardones, Juan S BonifacinoAbstract:The biosynthetic sorting of Acid Hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the Acid Hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.
Grazyna Biala - One of the best experts on this subject based on the ideXlab platform.
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Effects of Fatty Acid Amide Hydrolase Inhibitors Acute Administration on the Positive and Cognitive Symptoms of Schizophrenia in Mice
Molecular Neurobiology, 2019Co-Authors: Marta Kruk-slomka, Izabela Banaszkiewicz, Tomasz Slomka, Grazyna BialaAbstract:The connection between the endocannabinoid system (ECS) and schizophrenia is supported by a large body of research. The ECS is composed of two types cannabinoid (CB: CB1 and CB2) receptors and their endogenous ligands, endocannabinoids. The best-known endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are intracellularly degraded by fatty Acid Hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Thus, the function of ECS might be modulated in a direct way, through CB receptor ligands or indirectly by FAAH and MAGL inhibitors. We evaluated that the direct influence of ECS, using FAAH (URB 597) and MAGL (JZL 184) inhibitors, on the schizophrenia-like effects in mice. The behavioral schizophrenia-like symptoms were obtained in animals by using N-methyl D-aspartate (NMDA) receptor antagonists, MK-801. An acute administration of MK-801 (0.3 and 0.6 mg/kg) induced psychotic symptoms in rodents, manifested as the increase in locomotor activity, measured in actimeters, as well as the memory impairment, assessed in the passive avoidance (PA) task. We revealed that an acute administration of URB 597, at the dose of 0.3 mg/kg, attenuated MK-801 (0.6 mg/kg)-induced memory impairment. In turn, an acute administration of URB 597 at a higher dose (1 mg/kg) potentiated MK-801 (0.3 mg/kg)-induced memory impairment. Similarly, an acute administration of JZL 184 (20 and 40 mg/kg) intensified an amnestic effect of MK-801 (0.3 mg/kg). Moreover, an acute injection of JZL 184 (1 mg/kg) potentiated hyperlocomotion is provoked by MK-801 (0.3 and 0.6 mg/kg) administration. The present findings clearly indicate that ECS, through an indirect manner, modulates a variety of schizophrenia-like responses in mice.
