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)

1. The Brown Norway (B/N) Katholiek rat is a mutant strain of plasma kininogen deficiency. The plasma of B/N-Katholiek rats was shown to contain only 3-5% of high-molecular-weight and low-molecular-weight kininogens (HK and LK) of the normal level by specific RIA, and 30% of prekallikrein was detected by amidase activity. However, HK antigen in the liver microsomal fraction of B/N-Katholiek rats was about 60% of that of normal rats. 2. In this paper we compare and discuss synthesis and secretion of HK and LK by primary cultures of livers of deficient and normal rats. The deficient hepatocytes could synthesize HK and LK in the same way as normal cells but could not secrete mature forms of HK and LK in the medium. Examination of the subcellular localization of the mutant HK in the hepatocytes showed that a larger amount of mutant HK antigen, compared to normal rats, was found in the 10,000 g fraction, which is rich in lysosomes, suggesting that the mutant HK may be transported to the lysosomes. 3. We also analyzed sequence of the HK cDNA of B/N-Katholiek and B/N-Kitasato rats and found a point mutation of G to A at nucleotide 487, which locates at the heavy chain region of HK and LK. 4. We constructed five expression plasmids to transfect COS-1 cells to examine HK secretion. COS-1 cells transfected with the plasmids containing the G to A transition could not secrete and retained HK, while those cells transfected with the plasmids containing normal G released HK into the medium. 5. These results indicate that a point mutation G to A at nucleotide 487, resulting in an amino acid transition from alanine (163) to threonine, is responsible for the defective secretion of HK and LK by the liver of B/N-Katholiek rats. We also discuss other cases of secretion defect of plasma proteins reported in the literature.
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PMID:Molecular mechanism of kininogen deficiency in brown Norway Katholiek rats. 774 70

Penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own alpha-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.
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PMID:Penicillin acylase has a single-amino-acid catalytic centre. 781 45

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
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PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

The CHA1 gene of Saccharomyces cerevisiae encodes the catabolic L-serine (L-threonine) deaminase responsible for the utilization of serine/threonine as nitrogen sources. Previously, we identified two serine/threonine response elements in the CHA1 promoter, UASCHA. We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHA1 transcription. The cha4-1 allele causes noninducibility of a CHA1 p-lacZ translational gene fusion, indicating that Cha4p exerts its action through the CHA1 promoter. Molecular and genetic mapping positioned the cha4 locus 17 cM centromere proximal to put1 on chromosome XII. The coding region of CHA4 predicts a 648-amino acid protein with a DNA-binding motif (residues 43-70) belonging to the Cys6 zinc cluster class. Gel retardation employing a recombinant peptide, Cha4p1-174, demonstrated that the peptide in vitro specifically binds UASCHA. Binding is abolished by a G-C to T-A mutation in the middle bases of the two CEZ-elements in UASCHA. The transcriptional activating ability of UASCHA derivatives in vivo correlates with their ability to bind Cha4p1-174 in vitro. We conclude that Cha4p is a positive regulator of CHA1 transcription and that Cha4p alone, or as part of a complex, is binding UASCHA.
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PMID:Cha4p of Saccharomyces cerevisiae activates transcription via serine/threonine response elements. 888 13

Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates. Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding. Asp-66 is a new residue found to be important in enzyme activity. The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntn-amidohydrolase family of enzymes.
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PMID:Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase. 954 3

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments. Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme [Altamirano et al. (1994) Eur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket [Oliva et al. (1995) Structure 3, 1323-1332]. Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects. Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosamine 6-P deaminase.
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PMID:Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase. 960 Oct 45

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.
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PMID:Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase. 980 67

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.
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PMID:Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides. 1020 83

The AmiC protein in Pseudomonas aeruginosa is the negative regulator and ligand receptor for an amide-inducible aliphatic amidase operon. In the wild-type PAC1 strain, amidase expression is induced by acetamide or lactamide, but not by butyramide. A mutant strain of P. aeruginosa, PAC181, was selected for its sensitivity to induction by butyramide. The molecular basis for the butyramide inducible phenotype of P.aeruginosa PAC181 has now been determined, and results from a Thr-->Asn mutation at position 106 in PAC181-AmiC. In the wild-type PAC1-AmiC protein this residue forms part of the side wall of the amide-binding pocket but does not interact with the acetamide ligand directly. In the crystal structure of PAC181-AmiC complexed with butyramide, the Thr-->Asn mutation increases the size of the ligand binding site such that the mutant protein is able to close into its 'on' configuration even in the presence of butyramide. Although the mutation allows butyramide to be recognized as an inducer of amidase expression, the mutation is structurally sub-optimal, and produces a significant decrease in the stability of the mutant protein.
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PMID:Structural adaptation to selective pressure for altered ligand specificity in the Pseudomonas aeruginosa amide receptor, amiC. 1070 52

In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.
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PMID:Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering. 1086 83


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