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)

Penicillin G acylase from Escherichia coli ATCC 11105 is synthesized from its precursor polypeptide into a catalytically active heterodimer via a complex posttranslational processing pathway. Substitutions in the pair of aminoacyl residues at the cleavage site for processing the small and large subunits were made. Their processing phenotypes and penicillin G acylase activities were analyzed. By the introduction of a prolyl residue at either position, the processing of the small subunit was blocked without a change in enzymatic activity. Four other substitutions had no effect. At the site for processing the large subunit, four substitutions out of the seven examined blocked processing. In general, penicillin G acylase activity seemed to be proportional to the efficiency of the large-subunit-processing step. Ser-290 is an amino acid critical for processing and also for the enzymatic activity of penicillin G acylase. In the mutant pAATC, in which Ser-290 is mutated to Cys, the precursor is processed, but there is no detectable enzymatic activity. This suggests that there is a difference in the structural requirements for the processing pathway and for enzymatic activity. Recombination analysis of several mutants demonstrated that the small subunit can be processed only when the large subunit is processed first. Some site-directed mutants from which signal peptides were removed showed partial processing phenotypes and reduced enzymatic activities. Their expression showed that the prerequisite for penicillin G acylase activity is the efficient processing of the large subunit and that the maturation of the small subunit does not affect the enzymatic activity.
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PMID:Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105. 140 Jan 78

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the alpha- and beta-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of alpha-subunit linked to a 54-amino-acid endopeptide and beta-subunit was lower than with the subunits alone.
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PMID:Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits. 155 6

The nucleotide sequence of the gene (pac) coding for penicillin G acylase from E. coli ATCC 11105 was determined and correlated with the primary structure of the two constituent subunits of this enzyme. The pac gene open reading frame consists of four structural domains: Nucleotide positions 1-78 coding for a signal peptide, positions 79-705 coding for the alpha subunit, positions 706-867 coding for a spacer peptide, and positions 868-2538 coding for the beta subunit. Plasmids were constructed which direct the synthesis of a pac gene product lacking the signal peptide, and the synthesis of the alpha subunit or the beta subunit. The following results were obtained: The two dissimilar subunits are processing products of a single precursor polypeptide; the spacer peptide is removed during processing; the precursor polypeptide lacking the signal sequence is accumulated in the cytoplasm; it is not processed proteolytically in the cytoplasm and it does not display enzyme activity. Processing, therefore, requires translocation through the cytoplasmic membrane; processing follows a distinct sequential pathway in vitro.
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PMID:Penicillin acylase from E. coli: unique gene-protein relation. 301 63

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
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PMID:Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli penicillin G amidase precursor polypeptide. 776 24

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydrolase involved in the degradation of glycoproteins. The synthesized precursor polypeptide of AGA is rapidly activated in the endoplasmic reticulum by proteolysis into two subunits. Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independently folded subunits totally lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer of the enzyme. Both of the subunits are required for the enzyme activity, and the immediate interaction of the subunits in the endoplasmic reticulum is necessary for the correct folding of the dimeric enzyme molecule. The specific amino acid residues essential for the active site of the AGA enzyme were further analyzed by site-directed mutagenesis and in vitro expression of mutagenized constructs. Replacement of Thr206, the most amino-terminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular processing or transport of the mutant polypeptide to the lysosomes. Analogously, replacement of the most amino-terminal tryptophan, Trp34 with Phe or Ser in the alpha-subunit, resulted in a totally inactive enzyme without influencing the intracellular processing or stability of the polypeptide. These results suggest that the catalytic center of this amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp34 in the alpha-subunit and Thr206 in the beta-subunit.
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PMID:Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase. 787 64

Various concentrations of isopropyl beta-D-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recombinant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacIq. At low IPTG concentrations (0.025-0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
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PMID:Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase. 916 51

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-amino cephalosporanic acid (GL-7ACA) to produce 7-amino cephalosporanic acid (7-ACA). The acylase from Pseudomonas sp. 130 (CA-130) is highly active on GL-7ACA and glutaryl 7-aminodesacetoxycephalosporanic acid (GL-7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by alpha- and beta-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the beta-subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N-terminal nucleophile hydrolases) described recently.
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PMID:In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130. 1041 32

Glutaryl-7-aminocephalosporanicacid acylase from Pseudomonas sp. GK16 produces glutaryl-7-aminocephalosporanic acid, a key intermediate for the synthesis of cephem antibiotics. Sequence alignment suggests that the enzyme may belong to the N-terminal nucleophile hydrolase superfamily including penicillin G acylase. The enzyme is an (alphabeta)(2) heterotetramer of two nonidentical subunits. These subunits are derived from a nascent precursor polypeptide that is cleaved proteolytically through a two-step autocatalytic process upon folding. The enzyme has been crystallized using the vapor diffusion method. A bipyramidal crystal form was obtained from a solution containing polyethylene glycol (MW 3350) and calcium chloride. Complete diffraction data sets have been collected up to 2.8 A resolution. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit cell parameters are a = b = 73.5 A, c = 380.3 A. Considerations of the possible values of V(m) account for the presence of a tetramer in the asymmetric unit.
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PMID:Crystallization and preliminary X-Ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16. 1094 72

Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (alphabeta)(2) heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free alpha-amino group of the N-terminal Ser-199 of the beta subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pK(a) = 6.0, which could be attributed to the alpha-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the alpha-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own alpha-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.
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PMID:The role of alpha-amino group of the N-terminal serine of beta subunit for enzyme catalysis and autoproteolytic activation of glutaryl 7-aminocephalosporanic acid acylase. 1099 36

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains of the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond between the C-terminal residue of the spacer and the active-site serine residue at the N terminus of the B chain. We have determined the crystal structure of a slowly processing precursor mutant (Thr263Gly) of penicillin G acylase from Escherichia coli, which reveals that the spacer peptide blocks the entrance to the active-site cleft consistent with an autocatalytic mechanism of maturation. In this mutant precursor there is, however, an unexpected cleavage at a site four residues from the active-site serine residue. Analyses of the stereochemistry of the 260-261 bond seen to be cleaved in this precursor structure and of the 263-264 peptide bond have suggested factors that may govern the autocatalytic mechanism.
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PMID:Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft. 1099 30


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