scholarly journals Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp. Strain KM

2003 ◽  
Vol 69 (7) ◽  
pp. 3849-3857 ◽  
Author(s):  
Takako Mukai ◽  
Shigeyuki Kawai ◽  
Hirokazu Matsukawa ◽  
Yuhsi Matuo ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Nad Kinase ◽  
Inorganic Polyphosphate ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K m values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

Cloning and Nucleotide Sequencing of aStaphylococcus aureus Gene Encoding a Branched-Chain-Amino-Acid Transporter

1998 ◽  
Vol 64 (2) ◽  
pp. 763-767 ◽  
Author(s):  
Uriwan Vijaranakul ◽  
Anming Xiong ◽  
Katherine Lockwood ◽  
R. K. Jayaswal
Keyword(s):  
Amino Acid ◽  
Insertion Site ◽  
Amino Acid Sequences ◽  
Nucleotide Sequencing ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureuschromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

scholarly journals Molecular Identification of Family 38 α-Mannosidase of Bacillus sp. Strain GL1, Responsible for Complete Depolymerization of Xanthan

2002 ◽  
Vol 68 (6) ◽  
pp. 2731-2736 ◽  
Author(s):  
Hirokazu Nankai ◽  
Wataru Hashimoto ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Reading Frame ◽  
A Cell ◽  
Terminal Amino ◽  
Amino Acid Sequence Information

ABSTRACT When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, α-mannosidase exhibiting activity toward p-nitrophenyl-α-d-mannopyranoside (pNP-α-d-Man) was produced intracellularly. The 350-kDa α-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-α-d-Man (Km = 0.49 mM) and d-mannosyl-(α-1,3)-d-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for α-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of α-mannosidases belonging to glycoside hydrolase family 38.

scholarly journals Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

2010 ◽  
Vol 76 (17) ◽  
pp. 5892-5901 ◽  
Author(s):  
Yoshikazu Shimada ◽  
Setsuko Yasuda ◽  
Masayuki Takahashi ◽  
Takashi Hayashi ◽  
Norihiro Miyazawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enteric Bacteria ◽  
Amino Acid Sequences ◽  
Soy Isoflavones ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Binding Motifs ◽  
Reading Frame ◽  
Cofactor Binding ◽  
International Patent

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

scholarly journals Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide

1996 ◽  
Vol 316 (2) ◽  
pp. 685-690 ◽  
Author(s):  
Masahiro TAMOI ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Higher Plants ◽  
Sequence Similarity ◽  
Native Enzyme ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
Synechococcus Pcc 7942 ◽  
Pcc 7942

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62±4.5 and 420±10.5 μM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx. molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

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

2007 ◽  
Vol 75 (11) ◽  
pp. 5255-5263 ◽  
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.

scholarly journals Bombyx mori Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA

1998 ◽  
Vol 72 (4) ◽  
pp. 3107-3116 ◽  
Author(s):  
Victor S. Mikhailov ◽  
Alla L. Mikhailova ◽  
Masashi Iwanaga ◽  
Sumiko Gomi ◽  
Susumu Maeda
Keyword(s):  
Dna Binding ◽  
Molecular Mass ◽  
Bombyx Mori ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Amino Acid Sequences ◽  
Single Stranded Dna ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived fromBombyx mori) infected with the B. morinucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180 ). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

scholarly journals Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus3001

1999 ◽  
Vol 181 (21) ◽  
pp. 6642-6649 ◽  
Author(s):  
Jae Kweon Park ◽  
Kumiko Shimono ◽  
Nobuhisa Ochiai ◽  
Kazutaka Shigeru ◽  
Masako Kurita ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Glycol Chitosan ◽  
Gene Encoding ◽  
Reading Frame ◽  
Acid Protein ◽  
Green Fluorescent ◽  
Potential Inhibitors

ABSTRACT The extracellular chitosanase (34,000 M r) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

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