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

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.

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Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein

Journal of Cell Science ◽

10.1242/jcs.110.12.1351 ◽

1997 ◽

Vol 110 (12) ◽

pp. 1351-1359

Author(s):

P. Castagnola ◽

M. Gennari ◽

R. Morello ◽

L. Tonachini ◽

O. Marin ◽

...

Keyword(s):

Chick Embryo ◽

Developmentally Regulated ◽

Reading Frame ◽

Amino Terminal ◽

Nuclear Antigens ◽

Cartilage Extracellular Matrix ◽

Low Levels ◽

Specific Rabbit ◽

Chicken Protein

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).

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Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

Applied and Environmental Microbiology ◽

10.1128/aem.64.2.549-554.1998 ◽

1998 ◽

Vol 64 (2) ◽

pp. 549-554 ◽

Cited By ~ 26

Author(s):

Ji-Quan Liu ◽

Saeko Ito ◽

Tohru Dairi ◽

Nobuya Itoh ◽

Michihiko Kataoka ◽

...

Keyword(s):

Amino Acid ◽

Significant Loss ◽

Site Directed Mutagenesis ◽

Nucleotide Sequencing ◽

Predicted Amino Acid Sequence ◽

Amino Acid Residues ◽

Purification And Characterization ◽

Reading Frame ◽

Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

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Purification and characterization of Haemophilus influenzae pili, and their structural and serological relatedness to Escherichia coli P and mannose-sensitive pili.

Journal of Experimental Medicine ◽

10.1084/jem.161.1.145 ◽

1985 ◽

Vol 161 (1) ◽

pp. 145-159 ◽

Cited By ~ 13

Author(s):

N G Guerina ◽

S Langermann ◽

G K Schoolnik ◽

T W Kessler ◽

D A Goldmann

Keyword(s):

Haemophilus Influenzae ◽

Cross Reactivity ◽

Coli Strain ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Cell Agglutination ◽

E Coli ◽

Amino Terminal ◽

Multiple Copies

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

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Features of the cellodextrinase gene from Fibrobacter succinogenes S85

Canadian Journal of Microbiology ◽

10.1139/m94-094 ◽

1994 ◽

Vol 40 (7) ◽

pp. 592-596 ◽

Cited By ~ 15

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.

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Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

Infection and Immunity ◽

10.1128/iai.68.8.4539-4548.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4539-4548 ◽

Cited By ~ 58

Author(s):

M. D. Glew ◽

L. Papazisi ◽

F. Poumarat ◽

D. Bergonier ◽

R. Rosengarten ◽

...

Keyword(s):

Phase Variation ◽

Gene Families ◽

Surface Proteins ◽

Dna Rearrangements ◽

Reading Frame ◽

Mycoplasma Agalactiae ◽

Amino Terminal ◽

Clonal Variant ◽

Variable Surface

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

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Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1017-1025.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1017-1025

Author(s):

Z Hong ◽

P Mann ◽

N H Brown ◽

L E Tran ◽

K J Shaw ◽

...

Keyword(s):

Cell Wall ◽

Killer Toxin ◽

Structural Defects ◽

Beta Glucan ◽

Calculated Molecular Mass ◽

Beta Glucanase ◽

Reading Frame ◽

Glucan Synthesis ◽

The Right

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

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

Journal of Bacteriology ◽

10.1128/jb.184.9.2399-2403.2002 ◽

2002 ◽

Vol 184 (9) ◽

pp. 2399-2403 ◽

Cited By ~ 30

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.

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

Biochemical Journal ◽

10.1042/bj3160057 ◽

1996 ◽

Vol 316 (1) ◽

pp. 57-63 ◽

Cited By ~ 35

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.

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Characterization of an Immunogenic Outer Membrane Autotransporter Protein, Arp, of Bartonella henselae

Infection and Immunity ◽

10.1128/iai.00533-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5255-5263 ◽

Cited By ~ 12

Author(s):

Christine M. Litwin ◽

Mindy L. Rawlins ◽

Erica M. Swenson

Keyword(s):

Amino Acid ◽

Molecular Mass ◽

Outer Membrane ◽

Dna Sequences ◽

Bartonella Henselae ◽

Cross Reactivity ◽

Passenger Domain ◽

Calculated Molecular Mass ◽

Reading Frame

ABSTRACT Bartonella henselae is a recently recognized pathogenic bacterium associated with cat scratch disease, bacillary angiomatosis, and bacillary peliosis. This study describes the cloning, sequencing, and characterization of an antigenic autotransporter gene from B. henselae. A cloned 6.0-kb BclI-EcoRI DNA fragment expresses a 120-kDa B. henselae protein immunoreactive with 21.2% of sera from patients positive for B. henselae immunoglobulin G antibodies by indirect immunofluorescence, with 97.3% specificity and no cross-reactivity with antibodies against various other organisms. DNA sequencing of the clone revealed one open reading frame of 4,320 bp with a deduced amino acid sequence that shows homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane and consist of a passenger region, the α-domain, and an outer membrane transporter region, the β-domain. The passenger domain shows homology to a family of pertactin-like adhesion proteins and contains seven, nearly identical 48-amino-acid repeats not found in any other bacterial or Bartonella DNA sequences. The passenger α-domain has a calculated molecular mass of 117 kDa, and the transporter β-domain has a calculated molecular mass of 36 kDa. The clone expresses a 120-kDa protein and a protein that migrates at approximately 38 kDa exclusively in the outer membrane protein fraction, suggesting that the 120-kDa passenger protein remains associated with the outer membrane after cleavage from the 36-kDa transporter.

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