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)

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
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PMID:Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis. 3 10

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea. In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
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PMID:Protease I from Escherichia coli. Some physicochemical properties and substrate specificity. 79 43

Further properties of cow liver deaminase are reported. Highly purified deaminase migrated as a single badn on starch and polycrylamide gels electrophoresis. Molecular weight determinations by means of gel filtration on calibrated columns of Sephadex G-100, Sepharose 4 B and B 10-Gel P-100, gave values of 40 000 +/- 4 000. Data obtained suggest that cow liver deaminase exists as a single polypeptide chain. Heating partially purified preparations of deminase resulted in an enhancement of activity. Added cosynthetase to these fractions increased the percentage of uroporphyrinogen III formed but also diminished total uroporphyrinogens synthesis. The action of several compounds added to the system was studied. Thiol reagents and divalent metals as Hg 2+ , Pb2+, (d2+ and Zn2+ inhibited deaminase, indicating the presence of thiol groups essential for activity, probably involved in the cyclization step. Certain concentrations of sodium, potassium and magnesium salts enhanced activity. Several chelators tested were without effect on the deminase. Some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the enzyme.
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PMID:Studies on cow liver porphobilinogen deaminase. 105 1

The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spoIID. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28-7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.
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PMID:Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. 135 79

Aculeacin A acylase (AAC), produced by Actinoplanes utahensis, catalyzes the hydrolysis of the palmitoyl moiety of the antifungal antibiotic, aculeacin A. Using mixed oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequences of the two subunits of AAC, overlapping clones were identified in a cosmid library of A. utahensis DNA. After the sub-cloning of a 3.0-kb fragment into Streptomyces lividans, the recombinant produced AAC extracellularly. The nucleotide sequence of this fragment predicted an open reading frame of 2358 bp with GTG start and TGA stop codons. The deduced 786-aa sequence should correspond to a single polypeptide chain, indicating that this polypeptide is processed to the active form which is composed of the two subunits. Threefold more AAC was obtained from the S. lividans recombinant carrying the cloned gene than the original A. utahensis strain.
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PMID:Cloning and sequencing of the aculeacin A acylase-encoding gene from Actinoplanes utahensis and expression in Streptomyces lividans. 139 88

A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide.
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PMID:A 138-kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity. 154 93

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.
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PMID:Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase. 188 10

The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.
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PMID:Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties. 189 18

The mutation that causes a deficiency of the lysosomal amidase, glycosylasparaginase, has been characterized in fibroblasts from three Finnish patients diagnosed with aspartylglucosaminuria (AGU). The polymerase chain reaction was used to amplify the glycosylasparaginase protein coding sequence from the three AGU patients in order to compare them to the normal sequence from a full-length human placenta cDNA clone HPAsn.6 (Fisher, K.J., Tollersrud, O.K., and Aronson, N.N., Jr. (1990) FEBS Lett. 269, 440-444). Two base changes were found to be common to all three Finnish AGU patients, a G482----A transition that results in an Arg161----Gln substitution and a G488----C transversion that causes Cys163----Ser. Detection of both point mutations from PCR-amplified cDNA or genomic DNA was facilitated by their creation of new endonuclease restriction sites. Expression studies in COS-1 cells revealed only the Cys163----Ser mutation caused a deficiency of glycosylasparaginase activity. This same substitution also prevented the normal posttranslational processing of the precursor glycosylasparaginase polypeptide into its alpha and beta subunits. Cell-free expression of the single-chain glycosylasparaginase precusor did not produce an active enzyme, suggesting that post-translational generation of subunits may be required for catalytic activity.
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PMID:Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. Amino acid substitution Cys163----Ser abolishes the activity of lysosomal glycosylasparaginase and its conversion into subunits. 190 74

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.
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PMID:Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product. 190 62


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