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Lawrence P. Wackett - One of the best experts on this subject based on the ideXlab platform.
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Microbial biodegradation of Biuret: defining Biuret hydrolases within the isochorismatase superfamily
Environmental microbiology, 2018Co-Authors: Serina L. Robinson, Jonathan P. Badalamenti, Anthony G. Dodge, Lambros J. Tassoulas, Lawrence P. WackettAbstract:Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of Biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on Biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative Biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with Biuret in the range of 2-11 µmol min-1 mg-1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of Biuret. Importantly, it advances understanding of the microbial capacity for Biuret biodegradation in agricultural systems.
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New Family of Biuret Hydrolases Involved in s-Triazine Ring Metabolism
ACS Catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify, and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M–1 s–1and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate...
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A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism.
ACS catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for Biuret. Biuret hydrolase does not react with carboxyBiuret, the product of the enzyme immediately preceding Biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.
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Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth
Applied and environmental microbiology, 2006Co-Authors: Nir Shapir, Michael J Sadowsky, Gang Cheng, Lawrence P. WackettAbstract: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.
A. V. Kuznetsov - One of the best experts on this subject based on the ideXlab platform.
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Phase equilibria in binary subsystems of urea–Biuret–water system
Journal of Thermal Analysis and Calorimetry, 2010Co-Authors: T. S. Babkina, A. V. KuznetsovAbstract:Joint results of the differential scanning calorimetry (DSC) and thermogravimetry (TG) experiments were the basis for the fusion enthalpy and temperature determination of the Biuret (NH_2CO)_2NH (synthesis by-product of the urea fertilizer (NH_2)_2CO). Recommended values are Δ_m H = (26.1 ± 0.5) kJ mol^−1, T _m = (473.8 ± 0.4) K. The DSC method allowed for the phase diagrams of “water–Biuret,” “water–urea,” “urea–Biuret” binary systems to be studied; as a result, liquidus and solidus curves were precisely defined. Stoichiometry and decomposition temperature of the Biuret hydrate identified, composition of the compound in “urea–Biuret” system was suggested.
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phase equilibria in binary subsystems of urea Biuret water system
Journal of Thermal Analysis and Calorimetry, 2010Co-Authors: T. S. Babkina, A. V. KuznetsovAbstract:Joint results of the differential scanning calorimetry (DSC) and thermogravimetry (TG) experiments were the basis for the fusion enthalpy and temperature determination of the Biuret (NH2CO)2NH (synthesis by-product of the urea fertilizer (NH2)2CO). Recommended values are ΔmH = (26.1 ± 0.5) kJ mol−1, Tm = (473.8 ± 0.4) K. The DSC method allowed for the phase diagrams of “water–Biuret,” “water–urea,” “urea–Biuret” binary systems to be studied; as a result, liquidus and solidus curves were precisely defined. Stoichiometry and decomposition temperature of the Biuret hydrate identified, composition of the compound in “urea–Biuret” system was suggested.
T. S. Babkina - One of the best experts on this subject based on the ideXlab platform.
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Phase equilibria in binary subsystems of urea–Biuret–water system
Journal of Thermal Analysis and Calorimetry, 2010Co-Authors: T. S. Babkina, A. V. KuznetsovAbstract:Joint results of the differential scanning calorimetry (DSC) and thermogravimetry (TG) experiments were the basis for the fusion enthalpy and temperature determination of the Biuret (NH_2CO)_2NH (synthesis by-product of the urea fertilizer (NH_2)_2CO). Recommended values are Δ_m H = (26.1 ± 0.5) kJ mol^−1, T _m = (473.8 ± 0.4) K. The DSC method allowed for the phase diagrams of “water–Biuret,” “water–urea,” “urea–Biuret” binary systems to be studied; as a result, liquidus and solidus curves were precisely defined. Stoichiometry and decomposition temperature of the Biuret hydrate identified, composition of the compound in “urea–Biuret” system was suggested.
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phase equilibria in binary subsystems of urea Biuret water system
Journal of Thermal Analysis and Calorimetry, 2010Co-Authors: T. S. Babkina, A. V. KuznetsovAbstract:Joint results of the differential scanning calorimetry (DSC) and thermogravimetry (TG) experiments were the basis for the fusion enthalpy and temperature determination of the Biuret (NH2CO)2NH (synthesis by-product of the urea fertilizer (NH2)2CO). Recommended values are ΔmH = (26.1 ± 0.5) kJ mol−1, Tm = (473.8 ± 0.4) K. The DSC method allowed for the phase diagrams of “water–Biuret,” “water–urea,” “urea–Biuret” binary systems to be studied; as a result, liquidus and solidus curves were precisely defined. Stoichiometry and decomposition temperature of the Biuret hydrate identified, composition of the compound in “urea–Biuret” system was suggested.
Stephan M Cameron - One of the best experts on this subject based on the ideXlab platform.
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New Family of Biuret Hydrolases Involved in s-Triazine Ring Metabolism
ACS Catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify, and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M–1 s–1and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate...
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A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism.
ACS catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for Biuret. Biuret hydrolase does not react with carboxyBiuret, the product of the enzyme immediately preceding Biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.
Michael J Sadowsky - One of the best experts on this subject based on the ideXlab platform.
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New Family of Biuret Hydrolases Involved in s-Triazine Ring Metabolism
ACS Catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify, and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M–1 s–1and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate...
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A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism.
ACS catalysis, 2011Co-Authors: Stephan M Cameron, Katharina Durchschein, Jack E Richman, Michael J Sadowsky, Lawrence P. WackettAbstract:Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a Biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding Biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of Biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on Biuret physiologically. This is supported by the fact that of the 34 substrate analogs of Biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for Biuret. Biuret hydrolase does not react with carboxyBiuret, the product of the enzyme immediately preceding Biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.
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Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth
Applied and environmental microbiology, 2006Co-Authors: Nir Shapir, Michael J Sadowsky, Gang Cheng, Lawrence P. WackettAbstract: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.