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)

Formamidase from rat liver proved to be microheterogenous. After preparative isoelectric focusing in density gradient columns, two peaks of formamidase with identical substrate specificity were identified. By analytical focusing in thin layers of polyacrylamide or Sephadex G-75 SF, even five bands could be separated. Their isoelectric points were 4.75, 4.78, 4.82, 4.92 (main band) and 5.11, but their Michaelis constants did not differ significantly (54 to 62 mumol/l). An identical molecular weight of 34700 +/- 3200 for all bands was determined by disc electrophoresis. This value was confirmed by sedimentation analyses (so20,w = 3.00 S) and electrophoresis in the presence of sodium dodecyl-sulfate (Mr 34900 +/- 2300), which only gave a single band. The homogeneity was also confirmed by electrophoresis in the presence of 6M urea. Repeated disc electrophoresis of focusing under native conditions with single, isolated formamidases again resulted in different bands which were identified, not only by Coomassie Blue, but also by their hydrolytic cleavage of naphthyl acetate. Formamidase showed neither proteolytic nor asparagine-amidohydrolase activity and oligosaccharide conjugates were not detectable. Ampholytes, buffer ions, pH and peroxodisulfate did not affect the heterogeneity. "Initial burst" measurements with diethyl(4-nitrophenyl) phosphate yielded an equivalent weight of 36,300. Formylkynurenine reduced this inhibition very effectively. Thus, an extraordinary reactive serine residue appeared to be located in the catalytic site of formamidase. A participation of sulfhydrylgroups in the inactivating reaction of arsenite was excluded although two such groups were detected by 5,5'-dithiobis(2-nitrobenzoic acid). N-Bromosuccinimide reacted primarily with one of the nine tryptophan residues without loss of enzymatic activity, but a 18.6-fold excess of this reagent resulted in a complete loss of activity. The reaction rates of the most effective inhibitors and of the protective action of formylkynurenine were determined. Thus, formamidase must clearly be distinguished from typical serine esterases and proteases.
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PMID:[Formamidase--microheterogeneity, catalytic properties and inhibitors (author's transl)]. 8 81

A mutation in a gene designated gmdA has been found to lead to loss of ability of Aspergillus nidulans to use benzamide, phenylacetamide and several other amides as sole nitrogen sources for growth. The gmdA1 lesion results in low levels of an enzyme, called the general amidase, which has acitivity for a wide range of amide substrates. This enzyme is reressed by certain nitrogen-containing metabolites, including ammonium, but is probably not regulated by induction or by carbon catabolite repression. Evidence is presented for the general amidase being distinct from the previously characterized acetamidase and formamidase enzymes. The data also indicate that there is a fourth amidase capable of the hydrolysis of valeramide and hexanamide.
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PMID:Amide utilization in Aspergillus nidulans: evidence for a third amidase enzyme. 110 71

Aspergillus nidulans produces acetamidase and formamidase enzymes. The acetamidase is produced in reduced amounts during growth on glucose, whereas the formamidase is not greatly affected. Mutations in a gene, amdT, which affect glucose repression of amidases are described. One of these, amdT102, causes the acetamidase to be no longer subject to glucose repression and also affects ability to synthesize formamidase. The other, amdT19, results in both the formamidase and the acetamidase being subject to abnormally strong glucose repression, and also in increased maximal acetamidase activities. The dominance relationships at the amdT locus have been investigated. It is suggested that the amdT gene may play a positive role in controlling amidase synthesis.
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PMID:Mutants with altered glucose repression of amidase enzymes in Aspergillus nidulans. 455 22

A 3.2-kbp PstI fragment of DNA encoding formamidase from the methylotrophic bacterium Methylophilus methylotrophus which had previously been cloned (pNW3) [Wyborn, N.R., Scherr, D.J. & Jones, C.W. (1994) Microbiology 140, 191-195], was subcloned as a 2.3 kbp HindIII fragment (pNW323). Nucleotide sequencing showed that the subclone contained two genes which encoded formamidase (fmdA) and a possible regulatory protein (fmdB). Predicted molecular masses for FmdA and FmdB were 44438 Da (compared with approximately 44500 Da by electrospray mass spectrometry and 51000 Da by SDS/PAGE of the purified enzyme) and 12306 Da, respectively. The derived amino acid sequence of formamidase was supported by N-terminal amino acid sequencing of the enzyme and of proteolytic fragments prepared from it using V8 endoproteinase and was 57% similar to that of the acetamidase from Mycobacterium smegmatis. The structural similarities between these two enzymes, and their existence as a separate class of bacterial amidase, were confirmed by immunological investigations.
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PMID:Molecular characterisation of formamidase from Methylophilus methylotrophus. 884 93

We report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37746Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6-urease negative mutant, amidase activity was two- to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.
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PMID:Identification and characterization of an aliphatic amidase in Helicobacter pylori. 936 23

The ability to utilize formamide as a sole nitrogen source has been found in numerous fungi. We have cloned the fmdS gene encoding a formamidase from Aspergillus nidulans and found that it belongs to a highly conserved family of proteins separate from the major amidase families. The expression of fmdS is primarily regulated via AreA-mediated nitrogen metabolite repression and does not require the addition of exogenous inducer. Consistent with this, deletion analysis of the 5' region of fmdS has confirmed the presence of multiple AreA-binding sites containing a characteristic core GATA sequence. Under carbon starvation conditions the response to nitrogen starvation is eliminated, indicating that the lack of a carbon source may result in inactivation of AreA. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS with its transcript overlapping the fmdS coding region. Disruption of the 5' gene and analysis of the effects of overexpression of this gene on fmdS expression has shown that expression of this upstream gene interferes with fmdS transcription, resulting in a strong dependence on AreA activation for expression. Therefore the relative position of these two genes is essential for normal regulation of fmdS.
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PMID:The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene. 1113 96

The sequence has been determined of 80 888 bp of contiguous subtelomeric DNA, including the isp5 gene, from the right arm of chromosome I of Schizosaccharomyces pombe; 27 open reading frames (ORFs) longer than 100 codons are present, giving a density of one gene per 3.0 kb. Seven of the predicted proteins are members of the major facilitator superfamily (MFS) of transport proteins, including four amino acid permease homologues, bringing this family of amino acid permease sequences to 17 in Sz. pombe, and a phylogenetic analysis is presented. Also encoded is an allantoate permease homologue, a sulphate permease homologue and a probable urea active transporter. Predicted non-membrane proteins include a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), a class III aminotransferase, serine acetyltransferase, protein-L-isoaspartate O-methyltransferase, alpha-glucosidase, alpha-galactosidase, esterase/lipase, oxidoreductase of the short-chain dehydrogenase/reductase (SDR) family, aldehyde dehydrogenase, formamidase, amidase, flavohaemoprotein, a putative translation initiation inhibitor and a protein with similarity to a filamentous fungal conidiation-specific protein. The remaining six ORFs are likely to encode proteins, either because they have sequence similarity with hypothetical proteins or because they are known to be transcribed. Introns are scarce in the sequenced region: only three ORFs contain introns, with only one having multiple introns. The sequenced region also contains a single Tf1 transposon long terminal repeat (LTR). The sequence is derived from cosmid clones c869, c922 and c1039 and has been submitted to the EMBL database under entries SPAC869 (Accession No. AL132779), SPAC922 (AL133522) and SPAC1039 (AL133521).
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PMID:Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes. 1122 45

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.
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PMID:The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues. 1135 66

The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.
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PMID:Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor. 1249 81

Ammonia production is of great importance for the gastric pathogen Helicobacter pylori as a nitrogen source, as a compound protecting against gastric acidity, and as a cytotoxic molecule. In addition to urease, H. pylori possesses two aliphatic amidases responsible for ammonia production: AmiE, a classical amidase, and AmiF, a new type of formamidase. Both enzymes are part of a regulatory network consisting of nitrogen metabolism enzymes, including urease and arginase. We examined the role of the H. pylori amidases in vivo by testing the gastric colonization of mice with H. pylori SS1 strains carrying mutations in amiE and/or amiF and in coinfection experiments with wild-type and double mutant strains. A new cassette conferring resistance to gentamicin was used in addition to the kanamycin cassette to construct the double mutation in strain SS1. Our data indicate that the amidases are not essential for colonization of mice. The search for amiE and amiF genes in 53 H. pylori strains from different geographic origins indicated the presence of both genes in all these genomes. We tested for the presence of the amiE and amiF genes and for amidase and formamidase activities in eleven Helicobacter species. Among the gastric species, H. acinonychis possessed both amiE and amiF, H. felis carried only amiF, and H. mustelae was devoid of amidases. H. muridarum, which can colonize both mouse intestine and stomach, was the only enterohepatic species to contain amiE. Phylogenetic trees based upon the sequences of H. pylori amiE and amiF genes and their respective homologs from other organisms as well as the amidase gene distribution among Helicobacter species are strongly suggestive of amidase acquisition by horizontal gene transfer. Since amidases are found only in Helicobacter species able to colonize the stomach, their acquisition might be related to selective pressure in this particular gastric environment.
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PMID:Presence of active aliphatic amidases in Helicobacter species able to colonize the stomach. 1450 Apr 81


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