scholarly journals Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site

2000 ◽  
Vol 346 (3) ◽  
pp. 799-804 ◽  
Author(s):  
Wen-Yi WANG ◽  
Shwu-Huey LIAW ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Sequence Similarity ◽  
Distinct Group ◽  
Structural Data ◽  
Hydrolytic Activity ◽  
Degenerate Primers ◽  
Homologous Proteins ◽  
Reading Frame

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3ʹ and 5ʹ RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His211 in this conserved motif was shown to be very important in catalysis by site-specific modification with 14C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg2+ and Ca2+. The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

scholarly journals Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity

2002 ◽  
Vol 367 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Tiila-Riikka KIEMA ◽  
Jukka P. TASKINEN ◽  
Päivi L. PIRILÄ ◽  
Kari T. KOIVURANTA ◽  
Rik K. WIERENGA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Similarity ◽  
Structural Data ◽  
Homologous Proteins ◽  
Multifunctional Enzyme ◽  
Isomerase Activity ◽  
N Terminus ◽  
Partial Gene Duplication

Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of β-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3S)-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1—190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Δ3—Δ2-enoyl-CoA isomerase reactions. Domain B (residues 191—280) links domain A to the (3S)-dehydrogenase region, which includes both domain C (residues 281—474) and domain D (residues 480—583). Domains C and D carry features of the dinucleotide-binding and the dimerization domains of monofunctional HADs respectively. Domain E (residues 584—722) has sequence similarity to domain D of the perMFE-1, which suggests that it has evolved via partial gene duplication. Experiments with engineered perMFE-1 variants demonstrate that the H1/I competence of domain A requires stabilizing interactions with domains D and E. The variant His-perMFE (residues 288—479)Δ, in which the domain C is deleted, is stable and has hydratase-1 activity. It is proposed that the extreme C-terminal domain E in perMFE-1 serves the following three functions: (i) participation in the folding of the N-terminus into a functionally competent H1/I fold, (ii) stabilization of the dehydrogenation domains by interaction with the domain D and (iii) the targeting of the perMFE-1 to peroxisomes via its C-terminal tripeptide.

scholarly journals Protein structure and gene cloning of Syncephalastrum racemosum nuclease

1999 ◽  
Vol 339 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Heng-Chien HO ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Direct Sequencing ◽  
Coli Strain ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Computer Search ◽  
Degenerate Primers ◽  
Syncephalastrum Racemosum

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

scholarly journals Insulin regulation of a novel WD-40 repeat protein in adipocytes

2001 ◽  
Vol 168 (2) ◽  
pp. 325-332 ◽  
Author(s):  
BD Rodgers ◽  
MA Levine ◽  
M Bernier ◽  
C Montrose-Rafizadeh
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tissue Expression ◽  
Northern Blotting ◽  
Predicted Amino Acid Sequence ◽  
Mrna Levels ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Reading Frame

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

Molecular Characterization of a New Lectin from the Marine Alga Ulva pertusa

2004 ◽  
Vol 36 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Sheng Wang ◽  
Fu-Di Zhong ◽  
Yong-Jiang Zhang ◽  
Zu-Jian Wu ◽  
Qi-Ying Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Ulva Pertusa ◽  
Amino Acid Sequence Similarity ◽  
Degenerate Primers ◽  
Heat Stable ◽  
Rapid Amplification

Abstract A new lectin, named UPL1, was purified from a green alga Ulva pertusa by an affinity chromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectin was about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinating activity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. The lectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and had higher activity within pH 6–8. The N-terminal amino acid sequence of the purified lectin was determined (P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned by rapid amplification of cDNA ends (RACE) method (AY433960). Sequence analysis of upl1 indicated it was 1084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the mature UPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 amino acids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not show amino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigm of a novel lectin family.

scholarly journals Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

2006 ◽  
Vol 72 (3) ◽  
pp. 2206-2211 ◽  
Author(s):  
Meike Ballschmiter ◽  
Ole Fütterer ◽  
Wolfgang Liebl
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Thermotoga Maritima ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Amino Acid Sequence Similarity ◽  
Glycoside Hydrolase Family ◽  
Ph Optimum ◽  
Intracellular Enzyme ◽  
Hyperthermophilic Bacterium

ABSTRACT The gene for a novel α-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 α-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an α-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.

scholarly journals New Mg2+-Dependent Oxytetracycline Resistance Determinant Tet 34 in Vibrio Isolates from Marine Fish Intestinal Contents

2002 ◽  
Vol 46 (5) ◽  
pp. 1550-1552 ◽  
Author(s):  
Lisa Nonaka ◽  
Satoru Suzuki
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Open Reading Frame ◽  
Purine Nucleotide ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
Intestinal Contents ◽  
Seriola Quinqueradiata

ABSTRACT A new oxytetracycline (OTC) resistance (Otcr) determinant, Tet 34, was cloned from chromosomal DNA of Vibrio sp. no. 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata). The transformant, containing cloned Tet 34, could grow in broth containing 25 μg of drug per ml with 10 mM MgCl2. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis. Mg2+ binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg2+-dependent Otcr mechanism.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues
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