Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

One plausible hypothesis for selective neuronal death in sporadic amyotropic lateral sclerosis (ALS) is excitotoxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, which are a subtype of ionotropic glutamate receptors. The Ca2+ conductance of AMPA receptors differs markedly depending on whether the GluR2 (or GluR-B) subunit is a component of the receptor. The properties of GluR2 are generated posttranscriptionally by RNA editing at the Q/R site in the putative second membrane domain (M2), during which the glutamine (Q) codon is substituted by an arginine (R) codon. AMPA receptors containing the unedited form of GluR2Q have high Ca2+ permeability in contrast to the low Ca2+ conductance of those containing the edited form of GluR2R. The role of Ca(2+)-permeable AMPA receptors, particularly GluR2 Q/R site RNA editing status, in neuronal death has been clearly demonstrated both in mice deficient in editing at the GluR2 Q/R site and in mice transgenic for an artificial Ca(2+)-permeable GluR2 subunit. We analyzed the expression level of mRNA of each AMPA receptor subunit in individual motor neurons, as well as the editing efficiency of GluR2 mRNA at the Q/R site in the single neuron level in control subjects and ALS cases. There was no significant difference as to the expression profile of AMPA receptor subunits or the proportion of GluR2 mRNA to total GluRs mRNA between normal subjects and ALS cases. By contrast, the editing efficiency varied greatly, from 0% to 100%, among the motor neurons of each individual with ALS, and was not complete in 44 of them (56%), whereas it remained 100% in normal controls. In addition, GluR2 editing efficiency was more than 99% in the cerebellar Purkinje cells of ALS, spinocerebellar degeneration and normal control groups. Thus, GluR2 underediting occurs in a disease specific and region selective manner. GluR2 modification by RNA editing is a biologically crucial event for neuronal survival, and its deficiency is a direct cause of neuronal death. Therefore, marked reduction of RNA editing in ALS motor neurons may be a direct cause of the selective motor neuron death seen in ALS. It is likely that the molecular mechanism underlying the deficiency in RNA editing is a reduction in the activity of ADAR2, a double- strand RNA specific deaminase. The restoration of this enzyme activity in ALS motor neurons may open the novel strategy for specific ALS therapy.
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PMID:Deficient RNA editing of GluR2 and neuronal death in amyotropic lateral sclerosis. 1562 11

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.
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PMID:Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon. 1619 74

4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V(max) and a four-fold higher K(m) value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components.
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PMID:4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme. 1666 52

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.
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PMID:Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase. 1730 46

The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4 s(-1)/mM and 3059.3 s(-1)/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys(52), Ser(128), Ser(152)) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA(Asn) and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.
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PMID:The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln. 1732 42

To facilitate the easier production of D-amino acids using N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 degrees C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of D-amino acids.
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PMID:Improving the thermostability of N-carbamyl-D-amino acid amidohydrolase by error-prone PCR. 1898 37

The recent specialization for utilization of pesticides reported for Pseudomonas diminuta phosphotriesterase (pPTE) strongly suggests that this activity evolved from an enzyme endowed with promiscuous phosphotriesterase activity. Such a putative "generalist" enzyme was recently proposed to be a member of the new phoshotriesterase-like lactonase family (PLL). The promiscuous carboxylesterase and phosphodiesterase activities detected in pPTE and PLLs in turn paved the way for the prediction of the existence in nature of PTE-like enzymes with predominant carboxylesterase or phosphodiesterase activities. An "in silico" analysis of the related Mesorhizobium loti ORF MLL7664 and the biochemical characterization demonstrated its prominent carboxylesterase and low phosphotriesterase specificity. On the basis of sequence similarity with the phosphotriesterase homology protein from Escherichia coli and the carboxylesterase activity, we called it phosphotriesterase-like carboxylesterase (MloPLC). The carboxylesterase activity is strictly dependent on divalent cations, and as such MloPLC is the first phosphotriesterase-like metal-carboxylesterase characterized to date. In related enzymes of the amidohydrolase superfamily either glutamate or carboxylated lysine substitutes for MloPLC glutamate 183 and the residue appear invariantly involved in maintaining the structural integrity of the binuclear metal center. Accordingly, we changed Glu-183 to lysine or glutamine. All the tested activities were completely abolished in the E183Q mutant, while only a residual phosphotriesterase activity could be detected in the E183K mutant. Surprisingly, in the latter mutant a parallel 650-fold specificity increase in bis-p-nitrophenyl-phosphate (BpNP-P) was observed, turning MloPLC from a carboxylesterase into a phosphodiesterase. Chemical, structural, and kinetic data strongly suggested that K183 is not carboxylated and that the gain of the new function is assisted by the substrate.
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PMID:Evolution in the amidohydrolase superfamily: substrate-assisted gain of function in the E183K mutant of a phosphotriesterase-like metal-carboxylesterase. 1943 55

omega-Amidase (omega-amidodicarboxylate amidohydrolase, EC 3.5.1.3) isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase, and ester hydrolysis reactions. omega-Amidase activity toward alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria, and fungi. Despite its versatility, widespread occurrence, and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (alpha-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing alpha-ketoglutaramate and an assay procedure that measures alpha-ketoglutarate formation from alpha-ketoglutaramate in a 96-well plate format. We also describe a 96-well plate assay procedure that measures omega-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophotometrically with negligible background interference. The two assay procedures (hydrolysis of alpha-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying omega-amidase approximately 3600-fold from rat liver cytosol. The ratio of alpha-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. omega-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help to characterize the function of omega-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of omega-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.
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PMID:Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine. 1946 48

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.
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PMID:Identification of the putative tumor suppressor Nit2 as omega-amidase, an enzyme metabolically linked to glutamine and asparagine transamination. 1959 34

We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472-amino acid polypeptide generated a compact mixed alpha/beta fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNA(Gln) amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes, the specific activity of NylA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd. However, NylA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of the inactive complex between the activity-deficient S174A mutant of NylA and Acd, performed at 1.8 A resolution, suggested the following enzyme/substrate interactions: a Ser(174)-cis-Ser(150)-Lys(72) triad constitutes the catalytic center; the backbone N in Ala(171) and Ala(172) are involved in oxyanion stabilization; Cys(316)-S(gamma) forms a hydrogen bond with nitrogen (Acd-N(7)) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S150A, or K72A substitution in NylA by site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that Ser(174)-cis-Ser(150)-Lys(72) is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys(316) is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms and evolution of NylA in comparison with other Ser-reactive hydrolases.
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PMID:X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation. 1988 45


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