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Query: UNIPROT:P06889 (Mol)
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An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.
Comp Biochem Physiol B Biochem Mol Biol 2000 Nov
PMID:Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins. 1112 64

The N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) is used on an industrial scale for the production of D-amino acids. The crystal structure of D-NCAase was solved by multiple isomorphous replacement with anomalous scattering using xenon and gold derivatives, and refined to 1.95 A resolution, to an R-factor of 18.6 %. The crystal structure shows a four-layer alpha/beta fold with two six-stranded beta sheets packed on either side by two alpha helices. One exterior layer faces the solvent, whereas the other one is buried and involved in the tight intersubunit contacts. A long C-terminal fragment extends from a monomer to a site near a dyad axis, and associates with another monomer to form a small and hydrophobic cavity, where a xenon atom can bind. Site-directed mutagenesis of His129, His144 and His215 revealed strict geometric requirements of these conserved residues to maintain a stable conformation of a putative catalytic cleft. A region located within this cleft involving Cys172, Glu47, and Lys127 is proposed for D-NCAase catalysis and is similar to the Cys-Asp-Lys site of N-carbamoylsarcosine amidohydrolase. The homologous active-site framework of these enzymes with distinct structures suggests convergent evolution of a common catalytic mechanism.
J Mol Biol 2001 Feb 16
PMID:Crystal structure and site-directed mutagenesis studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft. 1123 98

Salivary gland products of haematophogous insects including tsetse flies (Diptera: Glossinidia) are involved in antihaemostasis to allow for efficient blood feeding. In addition, salivary products of tsetse are thought to indirectly support the metacyclogenesis and eventual transmission of the African trypanosome protozoan parasites to their mammalian hosts. We have previously characterized the major anticoagulant, Tsetse Thrombin Inhibitor (TTI), from salivary extracts, and described molecular aspects of its cDNA from a Glossina morsitans morsitans salivary gland cDNA library. In addition, a family of two related genes with growth factor and adenosine-deaminase motifs (TSGF-1 and TSGF-2) have also been described. Here, we report on the molecular aspects of three different cDNAs and their putative products expressed in salivary glands: cDNAs TAg5, Tsal1 and Tsal2. The full-length transcript encoded by Tsetse Antigen 5 (TAg5) cDNA is 926 bp excluding the poly(A) stretch, and has an open reading frame of 259 amino acids that can encode for a protein of 28 925 Da. The putative product of TAg5 shows extensive similarities to cDNAs characterized from Drosophila (Agr and Agr2) and sandfly Lutzomyia (LuLoAG5). The cDNAs Tsal1 and Tsal2 are predicted to encode for mature proteins of 45 612 Da (399 amino acids) and 43 930 Da (389 amino acids), respectively, and their putative products exhibit over 42% identity to one another. The N terminus of each putative protein contains a hydrophobic region with signal peptide characteristics indicating that they may be secretory in nature. Transcripts specific for TAg5 and Tsal2 genes can be detected in all developmental stages of tsetse while Tsal1 expression is limited to adult and larval stages. A reverse transcription polymerase chain reaction based amplification approach indicates that TAg5 transcipts can be detected from proventriculus and midgut tissues of the fly in addition to salivary glands, while Tsal1 and Tsal2 expression is restricted to salivary gland and proventriculus. The salivary glands of adult males are found to express higher levels of TAg5 and Tsal2 in comparison to females while no significant sex-based difference is observed for Tsal1 expression. The expression of these cDNAs in different tsetse species (G. m. morsitans, Glossina austeni and Glossina fuscipes) shows wide variations.
Insect Mol Biol 2001 Feb
PMID:Characterization of genes expressed in the salivary glands of the tsetse fly, Glossina morsitans morsitans. 1124 Jun 38

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.
Brain Res Mol Brain Res 2001 Mar 05
PMID:Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis. 1124 16

Adherence of pathogenic microorganisms to the cell surface is a key event during infection. We have previously reported the characterization of Listeria monocytogenes transposon mutants defective in adhesion to eukaryotic cells. One of these mutants had lost the ability to produce Ami, a 102 kDa autolytic amidase with an N-terminal catalytic domain and a C-terminal cell wall-anchoring domain made up of repeated modules containing the dipeptide GW ('GW modules'). We generated ami null mutations by plasmid insertion into L. monocytogenes strains lacking the invasion proteins InlA (EGDDeltainlA), InlB (EGDDeltainlB) or both (EGDDeltainlAB). These mutants were 5-10 times less adherent than their parental strains in various cell types. The adhesion capacity of the mutants was restored by complementation with a DNA fragment encoding the Ami cell wall-anchoring domain fused to the Ami signal peptide. The cell-binding activity of the Ami cell wall-anchoring domain was further demonstrated using the purified polypeptide. Growth of the ami null mutants constructed in EGD and EGDDeltainlAB backgrounds was attenuated in the livers of mice inoculated intravenously, indicating a role for Ami in L. monocytogenes virulence. Adhesive properties have recently been reported in the non-catalytic domain of two other autolysins, Staphylococcus epidermidis AtlE and Staphylococcus saprophyticus Aas. Interestingly, we found that these domains were also composed of repeated GW modules. Thus, certain autolysins appear to promote bacterial attachment by means of their GW repeat domains. These molecules may contribute to the colonization of host tissues by Gram-positive bacteria.
Mol Microbiol 2001 Mar
PMID:The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor. 1125 38

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.
Mol Microbiol 2001 May
PMID:The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues. 1135 66

Many bacteria express a surface-exposed proteinaceous layer, termed the S-layer, which forms a regular two-dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S-layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S-layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post-translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high-MW SLP shows weak homology to N-acetyl muramoyl-L-alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high-MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high-MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis to be transcribed.
Mol Microbiol 2001 Jun
PMID:Molecular characterization of the surface layer proteins from Clostridium difficile. 1140 22

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.
Mol Biotechnol 2001 Mar
PMID:Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability. 1143 8

N-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein cross-bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.
Mol Microbiol 2001 Jul
PMID:Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli. 1145 9

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.
J Mol Biol 2001 Oct 12
PMID:Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism. 1160 52


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