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)

Addition of oleoyl-CoA to incubations containing rat lung membranes and S-adenosyl [methyl-3H]methionine resulted in the formation of a previously unidentified nonpolar methylated lipid. The product was formed enzymatically, with an apparent Km for S-adenosylmethionine (AdoMet) of about 0.3 microM and half-maximal activity using about 0.1 mM oleoyl-CoA. Activity was highest in microsomes but present in other membranous fractions, including plasma membranes from mature human erythrocytes. Intact red blood cells formed the nonpolar methylated lipid intracellularly upon incubation with [methyl-3H]methionine and oleoyl-CoA. Product formation differed among membranes from various tissues. The nonpolar methylated lipid was analyzed by TLC, high performance liquid chromatography, and gas chromatography with radiodetection. It was identified as S-methyl-N-oleoylmercaptoethylamide by gas chromatography-mass spectrometry. Products obtained from oleoyl-CoA or palmitoyl-CoA, incubated with nonradioactive or [methyl-14C]AdoMet, were compared using electron impact and/or chemical ionization mass spectrometry. Inferred structures were confirmed using authentic standards. The methylated product was apparently formed by tissue as follows: (a) cleavage of oleoyl-CoA by an amidase to form S-oleoylmercaptoethylamine; (b) spontaneous rearrangement to form N-oleoylmercaptoethylamide; and (c) enzymatic methylation of the free thiol by AdoMet. Participation of the amidase was suggested by the biosynthesis of the amide (free thiol) using [1-14C]oleoyl-CoA.
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PMID:Biosynthesis of S-methyl-N-oleoylmercaptoethylamide from oleoyl coenzyme A and S-adenosylmethionine. 714 72

Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively. At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop. Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii. Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process. The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase. This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala). In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37. Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.
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PMID:A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA. 750 51

A recent report in this journal [Vairapandi, M. and Duker, N.J. (1993) Nucleic Acids Res. 21, 5323-5327) presented evidence of an activity in HeLa cell nuclear extracts that released radiolabeled material from a poly(dG.dC) polymer that had been methylated and simultaneously labeled on cytosine residues by incubation with a CpG-specific DNA methylase and [methyl-3H]S-adenosylmethionine. Based on chromatographic evidence that the released products were thymine and 5-methylcytosine and on f1p4olabeling data suggesting a concomitant increase in abasic sites, the authors concluded that the releasing activity was a 5-methylcytosine-specific glycosylase and that the solubilized 5-methylcytosine was converted to thymine by a nuclear deaminase. We have confirmed that HeLa nuclear extracts promote release of ethanol-soluble radioactivity from a methyl-labeled poly(dG-5-methyl-dC)polymer, but the products released were neither 5-methylcytosine nor thymine. Furthermore, free 5-methylcytosine was not deaminated by incubation with the nuclear extract. The labeled compound released initially from the polymer appeared to be 5-methyl-deoxycytidine monophosphate, which was converted to 5-methyl-deoxycytidine, thymidine monophosphate, and/or thymidine by further incubation with the nuclear extract. The activity responsible for the release, therefore, was a nuclease. Release of 32P-labeled nucleotides from a 32P-labeled poly(dG-dC) polymer suggested, furthermore, that the activity was not specific for methylated DNA.
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PMID:Enzymic removal of 5-methylcytosine from poly(dG-5-methyl-dC) by HeLa cell nuclear extracts is not by a DNA glycosylase. 778 19

The phenomenon of repeat-induced point mutation (RIP), acting during the sexual phase of the model eukaryote Neurospora crassa, is considered to study the putative in vivo relationships existing between cellular levels of S-adenosylmethionine (SAM), cytosine methylation and the occurrence of C-->T transition mutations. We analyse the kinetic behaviour of the different enzymatic models proposed to explain the underlying mutagenic mechanisms of RIP. The dependence of the mutation rate on the cellular levels of the methyl group donor SAM was evaluated for the models of mutation catalysed by a DNA-cytosine deaminase, a DNA-(5-methylcytosine) deaminase, a DNA-(5-cytosine) methyltransferase, and for a model combining the activities of the last two enzymes. We propose that these models can be distinguished by studying the dependence of RIP on intracellular SAM levels.
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PMID:Analysis of models involving enzymatic activities for the occurrence of C-->T transition mutations during repeat-induced point mutation (RIP) in Neurospora crassa. 962 39

5'-Fluoro-5'-deoxyinosine (5'-FDI) is identified as an adventitious side product that accumulates in cell free incubations of SAM and fluoride ion in Streptomyces cattleya. 5'-FDI was identified by a combination of isotopic labelling studies and co-synthesis studies as well as enzymatic degradation. Although it is an efficiently generated end product of the cell free incubations, 5'-FDI is not a biosynthetic intermediate and it does not accumulate as a fluorometabolite with fluoroacetate and 4-fluorothreonine in whole cell incubations of S. cattleya. Clearly the purine deaminase which converts 5'-fluoro-5'-deoxyadenosine (5'-FDA) to 5'-FDI in the cell free extract does not come into contact with 5'-FDA in whole cells, suggesting some level of compartmentalisation in cells of S. cattleya. The biotransformation of 5'-FDI from fluoride ion extends the range of organofluorine products, beyond biosynthetic intermediates, that can be generated by this system, for applications such as enzymatic labelling with fluorine-18 for positron emission tomography applications.
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PMID:The identification of 5'-fluoro-5-deoxyinosine as a shunt product in cell free extracts of Streptomyces cattleya. 1616 85

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine-sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III.
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PMID:A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase. 1673 88

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases, respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore, the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxobacteria is reported.
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PMID:Biochemical characterization of MelJ and MelK. 1691 25

Plant caffeic acid O-methyltransferases (COMTs) use S-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in vivo, and will generally utilize a range of phenolic compounds in vitro. Collazo et al. (Anal. Biochem. 2005, 342, 86-92) have published a discrete, end-point fluorescence assay to detect histone methyltransferases using S-adenosyl homocysteine hydrolase and adeonsine deaminase as coupling enzymes and a thiol-specific fluorophore, Thioglo1, as the detecting reagent. Using this previous assay as a guide, we have developed and validated a facile, sensitive and real-time fluorescence assay for characterizing plant COMTs and in the process simplified the original assay as well by obviating the need for adenosine deaminase in the assay, and simultaneously converting an end-point assay into a continuous one. Our assay has been used to kinetically characterize recombinant sorghum COMT (Bmr-12) a key enzyme involved in cell wall lignification, and analyze COMT activity in maturing tillers from switchgrass plants. Data indicated that the calculated K(m) and V(max) values for the recombinant sorghum COMT using different substrates in the fluorescent assay were similar to published values for COMT enzymes from other plant species. Native COMT activity was greatest in internodes at the top of a tiller and declined in the more basal internodes. This new assay should have broad applicability for characterizing COMTs and potentially other plant methlytransferases that utilize ado-met as a methyl donor.
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PMID:A continuous, quantitative fluorescent assay for plant caffeic acid O-methyltransferases. 2039 33

Blasticidin S is a peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes that exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesis in vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome of Streptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, for S. lividans blasticidin S deaminase) was identified in S. lividans and shown to govern this in vivo conversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin S in vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene in S. lividans LL2 led to successful production of active blasticidin S in the resultant mutant, S. lividans WJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster, blsE, blsF, and blsL, encoding a predicted radical S-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.
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PMID:Streptomyces lividans blasticidin S deaminase and its application in engineering a blasticidin S-producing strain for ease of genetic manipulation. 2337 31

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5'-amino-5'-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine ((m5)C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of (m5)C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung (+) host proficient in uracil excision repair.
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PMID:Cytosine-to-uracil deamination by SssI DNA methyltransferase. 2420 58


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