Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.
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PMID:Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme. 1245 Apr 10

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).
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PMID:Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation. 1247 34

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH 7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S0.5, parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that 'liver type' of AMP-deaminase is an enzyme form present in human term placenta.
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PMID:AMP-deaminase from human term placenta. 1457 11

AMP-deaminase from hen stomach smooth muscle was isolated and physico-chemical properties of the purified enzyme were investigated. The enzyme had an activity optimum at pH 6.5, and poorly deaminated the substrate analogues tested. At optimum pH (6.5), in the absence of regulatory ligands (control conditions), the enzyme manifested hyperbolic substrate-saturation kinetics with half-saturation constant (S(0.5)) of about 4.5 mM. Additions of adenine nucleotide effectors (ATP, ADP) activated the enzyme strongly at all the concentrations tested, diminishing significantly the value of S(0.5) constant. In contrast, the regulatory effect of orthophosphate was variable, and depended on the orthophosphate concentration used. The molecular mass of the enzyme subunit determined in SDS/PAG electrophoresis was about of 37kDa. The obtained results suggest that in different types of hen muscle, similarly as in humans and rats, expression of AMP-deaminase is under the control of independent genes.
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PMID:AMP-deaminase from hen stomach smooth muscle--physico-chemical properties of the enzyme. 1509 42

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.
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PMID:AMP-deaminase from normal and cirrhotic human liver: a comparative study. 1553 16

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.
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PMID:Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP. 1590 97

A Gram-positive bacterial strain able to degrade the herbicide atrazine was isolated using a simple model ecosystem constituted with Japanese riverbed sediment and its associated water (microcosm). Treatment of the water phase of the microcosm with 1 mg litre-1 [ring-14C]atrazine resulted in the rapid degradation of atrazine after a 10 day lag phase period. The [ring-14C]cyanuric acid formed was transiently accumulated as an intermediary metabolite in the water phase and was subsequently mineralised through triazine ring cleavage. Possible atrazine-degrading microbes suspended in the water phase of the microcosm were isolated by the plating method while rapid degradation of atrazine was in progress. Among the 48 strains that were isolated, 47 exhibited atrazine-degrading activity. From these 47 isolates, 12 strains that were randomly selected were found to identically convert atrazine to cyanuric acid via hydroxyatrazine. Polymerase chain reaction (PCR) amplification of the genes corresponding to atrazine degradation revealed that these strains at least carried the genes trzN (atrazine chlorohydrolase from Nocardioides C190) and atzC (N-isopropylammelide isopropyl amidohydrolase from Pseudomonas ADP). Physiological characteristics and 16S rDNA partial sequences of six strains that were further selected strongly suggested that all these isolates originated from the same Nocardioides sp. strain. Additionally, only one isolate could mineralise the triazine ring of cyanuric acid. Based on microscopic observations, this strain appears to be a two-membered microbial consortium consisting of Nocardioides sp. and a Gram-negative bacterium. In conclusion, atrazine biodegradation in the microcosm appeared to occur predominantly by Nocardioides sp. to yield cyanuric acid, which could be mineralised by the other relatively ubiquitous microbes.
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PMID:Characterisation of new strains of atrazine-degrading Nocardioides sp. isolated from Japanese riverbed sediment using naturally derived river ecosystem. 1649 96

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.
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PMID:Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth. 1659 48

Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The k(cat) and K(m) of AtzB with hydroxyatrazine as a substrate were 3 s(-1) and 20 microM, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.
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PMID:Hydroxyatrazine N-ethylaminohydrolase (AtzB): an amidohydrolase superfamily enzyme catalyzing deamination and dechlorination. 1766 Feb 79

AMP-deaminase was purified to electrophoretic homogeneity from white skeletal muscle of a teleost fish, the common carp, Cyprinus carpio. The purified enzyme was highly stable and showed non-Michaelis-Menten kinetics with a S(0.5) value for AMP of 2.52+/-0.16 mM (SEM) and a Hill coefficient of 1.19+/-0.11. Specific activity of the purified enzyme was 1000-1200 U/mg protein. The pH optimum was 6.3 and the enzyme was activated by ADP and ATP, but inhibited by phosphate and fluoride. Low concentrations of NaCl and KCl (100-150 mM) activated, whereas higher concentrations were inhibitory. Free radicals inactivated the enzyme, decreasing V(max) by one-half but not affecting S(0.5) or Hill coefficient. Possible regulatory mechanisms of AMP-deaminase activity in fish muscle are discussed.
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PMID:Regulation of AMP-deaminase activity from white muscle of common carp Cyprinus carpio. 1806 Aug 19


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