Features of the cellodextrinase gene from Fibrobacter succinogenes S85

1994 ◽  
Vol 40 (7) ◽  
pp. 592-596 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Gel Filtration ◽  
Fibrobacter Succinogenes ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Base Pairs ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Promoter Sequences ◽  
Putative Ribosome Binding Site ◽  
Coli Promoter

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.

scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

scholarly journals A novel tetrameric lectin from Lycoris aurea with four mannose binding sites per monomer.

2007 ◽  
Vol 54 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Jiwei Liu ◽  
Xiaochao Xu ◽  
Jinzhi Liu ◽  
Jan Balzarini ◽  
Yongtin Luo ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Sites ◽  
Tertiary Structure ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Hydrophobic Cluster Analysis ◽  
Mannose Binding ◽  
Narcissus Pseudonarcissus ◽  
Lycoris Aurea

The mannose-binding agglutinin from bulbs of Lycoris aurea (LAA) agglutinates rabbit but not human erythrocytes. The molecular mass of the monomer in SDS/PAGE is 12 kDa while the apparent molecular mass in gel filtration is 48 kDa, indicating that LAA is a homotetramer. The full-length cDNA of LAA contains 683 bp with an open reading frame encoding a protomer of 162 amino-acid residues. Hydrophobic Cluster Analysis and molecular modeling of the 109-residue mature polypeptide suggested a similar secondary and tertiary structure to those of Narcissus pseudonarcissus agglutinin (NPA). Molecular docking revealed that, besides the three mannose-binding sites common among Amaryllidaceae lectins, LAA also contains a fourth unique mannose-binding site formed by a tryptophan cluster. The existence of four mannose-binding sites in each monomer of LAA is very unusual and has only been reported for NPA earlier.

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

1999 ◽  
Vol 65 (7) ◽  
pp. 3001-3007 ◽  
Author(s):  
Frederic Chavagnat ◽  
Michael G. Casey ◽  
Jacques Meyer
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Streptococcus Thermophilus ◽  
Maximal Activity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
T7 Promoter ◽  
Reading Frame ◽  
Endopeptidase Activity

ABSTRACT The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.

scholarly journals Pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues

1996 ◽  
Vol 316 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Iris BRUCHHAUS ◽  
Thomas JACOBS ◽  
Martin DENART ◽  
Egbert TANNICH
Keyword(s):  
Entamoeba Histolytica ◽  
Prokaryotic Expression ◽  
Recombinant Expression ◽  
Expression System ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Prokaryotic Expression System ◽  
Competitive Inhibitors ◽  
Dna Fragment

By using oligonucleotide primers derived from regions highly conserved in prokaryotic and eukaryotic phosphofructokinase sequences, a genomic DNA fragment was amplified and used to isolate cDNA and genomic clones coding for PPi-dependent phosphofructokinase (PPi-PFK) of Entamoeba histolytica. The open reading frame consists of 1308 bp and the corresponding protein has a calculated molecular mass of 47.6 kDa. The N-terminal half of the protein shows 27–35% identity with PPi-PFKs or ATP-dependent phosphofructokinases (ATP-PFKs) of various eukaryotic and prokaryotic organisms. The amino acid residues that form the active site of the PPi-PFK from Propionibacterium freudenreichii and the allosteric ATP-PFK from Escherichia coli are conserved within the amoeba sequence. The PPi-PFK was recombinantly expressed by using a prokaryotic expression system. The purified recombinant protein was found to be enzymically active. The Km values for PPi and fructose 6-phosphate of the native and the recombinant PPi-PFKs were nearly identical. Various bisphosphonates (synthetic pyrophosphate analogues) were tested for their ability to inhibit PPi-PFK activity or amoebic growth. All bisphosphonates tested were competitive inhibitors for amoeba PPi-PFK activity. The best inhibitors were CGP 48048 and zoledronate, with Ki values of 50 μM. All bisphosphonates inhibited amoebic growth. One of them (risedronate) was inhibitory at a concentration of 10 μM. Bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.

scholarly journals Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis

1996 ◽  
Vol 318 (1) ◽  
pp. 157-162 ◽  
Author(s):  
Brunella PERITO ◽  
Nerino ALLOCATI ◽  
Enrico CASALONE ◽  
Michele MASULLI ◽  
Beatrice DRAGANI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteus Mirabilis ◽  
Burkholderia Cepacia ◽  
Glutathione Transferase ◽  
Amino Acid Identity ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Promoter Sequences ◽  
E Coli ◽  
Cloned Gene

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421–425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli σ70 consensus promoter and to promoters of P. mirabiliscat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside and growth at 37 °C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11

2007 ◽  
Vol 53 (2) ◽  
pp. 186-195 ◽  
Author(s):  
Kun Meng ◽  
Jiang Li ◽  
Yanan Cao ◽  
Pengjun Shi ◽  
Bo Wu ◽  
...  
Keyword(s):  
Protease Activity ◽  
Signal Sequence ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Proteinase K ◽  
Keratinolytic Activity ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Mature Enzyme ◽  
Phenylmethyl Sulfonyl Fluoride

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 °C for its caseinolytic activity and pH 9 and 62 °C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.

scholarly journals Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

2006 ◽  
Vol 72 (3) ◽  
pp. 1886-1890 ◽  
Author(s):  
Hyun Sook Lee ◽  
Yun Jae Kim ◽  
Seung Seob Bae ◽  
Jeong Ho Jeon ◽  
Jae Kyu Lim ◽  
...  
Keyword(s):  
Metal Binding ◽  
Genomic Analysis ◽  
Enzyme Characterization ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Hyperthermophilic Archaeon ◽  
Amino Terminal ◽  
Aminopeptidase P

ABSTRACT Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(N ε-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km , 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.

Molecular analysis of FMRFamide- and FMRFamide-related peptides (FaRPS) in the cuttlefish Sepia officinalis.

1997 ◽  
Vol 200 (10) ◽  
pp. 1483-1489 ◽  
Author(s):  
P K Loi ◽  
N Tublitz
Keyword(s):  
Basic Amino Acid ◽  
Sepia Officinalis ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Brain

The display of complex color patterns of the cuttlefish Sepia officinalis is under the regulation of the FMRFamide-related peptide (FaRP) family, but their exact identities are unknown. We report the isolation and characterization of a full-length FaRP cDNA from the brain of S. officinalis. This cDNA is 1850 base pairs long, including an open reading frame of 996 base pairs. The cDNA encodes a precursor protein containing four FaRPs: ALSGDAFLRF, FIRF, FLRF and FMRF. Each propeptide has a C-terminal glycine residue that is presumably converted post-translationally to an amide. Every FaRP propeptide is also flanked by basic amino acid residues at the amino and carboxy termini, indicative of putative cleavage sites during post-translational processing. Each of the four FaRPs encoded by this cDNA causes chromatophore expansion when assayed in an in vitro chromatophore bioassay. Thus, it is likely that one or more of the FaRPs identified in this study are involved in controlling chromatophore activity in cuttlefish.

scholarly journals The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

1990 ◽  
Vol 10 (5) ◽  
pp. 2214-2223 ◽  
Author(s):  
Y Wada ◽  
K Kitamoto ◽  
T Kanbe ◽  
K Tanaka ◽  
Y Anraku
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Lethal Event ◽  
Vacuolar Protein ◽  
And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function

1990 ◽  
Vol 10 (5) ◽  
pp. 2214-2223
Author(s):  
Y Wada ◽  
K Kitamoto ◽  
T Kanbe ◽  
K Tanaka ◽  
Y Anraku
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Lethal Event ◽  
Vacuolar Protein ◽  
And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

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