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.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

scholarly journals Cloning and Expression Analysis of a Farnesyl Diphosphate Synthase (FPPS) Gene from Chamaemelum nobile

2017 ◽  
Vol 45 (2) ◽  
pp. 358-364 ◽  
Author(s):  
Xiao-Meng LIU ◽  
Ting-Ting TAO ◽  
Xiang-Xiang MENG ◽  
Wei-Wei ZHANG ◽  
Jie CHANG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Condensation Reaction ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Farnesyl Diphosphate ◽  
Cloning And Expression ◽  
Farnesyl Diphosphate Synthase ◽  
Reading Frame ◽  
Chamaemelum Nobile ◽  
Multiple Alignment Analysis

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene (CnFPPS) was cloned from Chamaemelum nobile. The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile. Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla. The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile.

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 Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase

1998 ◽  
Vol 331 (3) ◽  
pp. 953-958 ◽  
Author(s):  
Hiro-omi TAMURA ◽  
Yuki HARADA ◽  
Atsushi MIYAWAKI ◽  
Katsuhiko MIKOSHIBA ◽  
Michio MATSUI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nasal Cavity ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Reading Frame ◽  
Residue Polypeptide

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).

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.

Structure of the Dictyostelium discoideum prestalk D11 gene and protein

1985 ◽  
Vol 5 (6) ◽  
pp. 1473-1479
Author(s):  
E Barklis ◽  
B Pontius ◽  
H F Lodish
Keyword(s):  
Amino Acid ◽  
Dictyostelium Discoideum ◽  
Sequence Data ◽  
Phosphodiesterase Inhibitor ◽  
Amino Acid Sequences ◽  
Base Pairs ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transcriptional Mapping ◽  
Initiator Codon

The gene encoding the prestalk D11 mRNA of Dictyostelium discoideum has been isolated and characterized. Transcriptional mapping and sequence data indicated that the D11 message is unspliced and contains an 846-base open reading frame. The 273 base pairs upstream from the translation initiator codon are 88% A + T, typical of Dictyostelium upstream sequences, and contain no recognizable upstream activator sequence. The deduced D11 protein is exceptionally rich in cysteine residues and consists of a 25-amino acid hydrophobic leader sequence (L) followed by a series of repeats with the structure LA1B1A2B2C1B3C2B4C3B5C4B6, where A, B, and C, are, respectively, amino acid sequences of 39, 18, and 15 residues. The deduced D11 protein shares certain similarities with the Dictyostelium cyclic AMP phosphodiesterase inhibitor protein.

scholarly journals Structure of the Dictyostelium discoideum prestalk D11 gene and protein.

1985 ◽  
Vol 5 (6) ◽  
pp. 1473-1479 ◽  
Author(s):  
E Barklis ◽  
B Pontius ◽  
H F Lodish
Keyword(s):  
Amino Acid ◽  
Dictyostelium Discoideum ◽  
Sequence Data ◽  
Phosphodiesterase Inhibitor ◽  
Amino Acid Sequences ◽  
Base Pairs ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transcriptional Mapping ◽  
Initiator Codon

The gene encoding the prestalk D11 mRNA of Dictyostelium discoideum has been isolated and characterized. Transcriptional mapping and sequence data indicated that the D11 message is unspliced and contains an 846-base open reading frame. The 273 base pairs upstream from the translation initiator codon are 88% A + T, typical of Dictyostelium upstream sequences, and contain no recognizable upstream activator sequence. The deduced D11 protein is exceptionally rich in cysteine residues and consists of a 25-amino acid hydrophobic leader sequence (L) followed by a series of repeats with the structure LA1B1A2B2C1B3C2B4C3B5C4B6, where A, B, and C, are, respectively, amino acid sequences of 39, 18, and 15 residues. The deduced D11 protein shares certain similarities with the Dictyostelium cyclic AMP phosphodiesterase inhibitor protein.

scholarly journals Identification and Characterization of thefis Operon in Enteric Bacteria

1998 ◽  
Vol 180 (22) ◽  
pp. 5932-5946 ◽  
Author(s):  
Michael B. Beach ◽  
Robert Osuna
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
E Coli

ABSTRACT The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, andProteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens,E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence forH. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) precedingfis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihfbinding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

Complete Genome Sequence of a Novel Ourmia-like Mycovirus Infecting the Phytopathogenic Fungus Botryosphaeria Dothidea

2021 ◽  
Author(s):  
Liying Sun ◽  
Ziqian Lian ◽  
Subha Das ◽  
Jingxian Luo ◽  
Ida Bagus Andika
Keyword(s):  
Amino Acid ◽  
Genome Sequence ◽  
Amino Acid Sequences ◽  
Shanxi Province ◽  
Phytopathogenic Fungus ◽  
Botryosphaeria Dothidea ◽  
Reading Frame ◽  
Ring Rot ◽  
Ssrna Virus ◽  
The Family

Abstract In this study, we describe the full-length genome sequence of a novel ourmia-like mycovirus, tentatively designated Botryosphaeria dothidea ourmia-like virus 1 (BdOLV1), isolated from the phytopathogenic fungus, Botryosphaeria dothidea strain P8, associated with apple ring rot in Shanxi province, China. The complete BdOLV1 genome is comprised of 2797 nucleotides, a positive-sense (+) single-stranded RNA (ssRNA) with a single open reading frame (ORF). The ORF putatively encodes a 642-amino acid polypeptide with conserved RNA-dependent RNA polymerase (RdRp) motifs, related to viruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp amino acid sequences showed that BdOLV1 is grouped with oomycete-infecting unclassified viruses closely related to the genus Botoulivirus in Botourmiaviridae. This is the first report of a novel (+)ssRNA virus in B. dothidea related to the genus Botoulivirus in the family Botourmiaviridae.

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