scholarly journals Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

1998 ◽  
Vol 329 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Katsuhiko FUKASAWA ◽  
M. Kayoko FUKASAWA ◽  
Makoto KANAI ◽  
Shingo FUJII ◽  
Junzo HIROSE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dipeptidyl Peptidase ◽  
Cloning And Expression ◽  
Zinc Binding ◽  
Binding Motif ◽  
Ph Optimum ◽  
Reading Frame ◽  
Theoretical Molecular Mass ◽  
Cloned Cdna ◽  
Zinc Binding Domain

We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-β-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-β-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7.4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-β-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

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 Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

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 Molecular cloning and functional characterization of the mouse organic-anion-transporting polypeptide 1 (Oatp1) and mapping of the gene to chromosome X

1999 ◽  
Vol 345 (1) ◽  
pp. 115-120 ◽  
Author(s):  
Bruno HAGENBUCH ◽  
Ilse-Dore ADLER ◽  
Thomas E. SCHMID
Keyword(s):  
Amino Acid ◽  
Organic Anion ◽  
Functional Characterization ◽  
Amino Acid Identity ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Reading Frame ◽  
Acid Identity ◽  
Organic Anion Transporting Polypeptide ◽  
Cloned Cdna

We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent Km values of 12 μM and 5 μM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.

scholarly journals Cloning, sequencing and expression of the pyrophosphate-dependent phosphofructo-1-kinase from Naegleria fowleri

1995 ◽  
Vol 307 (1) ◽  
pp. 143-149 ◽  
Author(s):  
K L Wessberg ◽  
S Skolnick ◽  
J Xu ◽  
F Marciano-Cabral ◽  
R G Kemp
Keyword(s):  
Nucleotide Binding ◽  
Binding Motif ◽  
Naegleria Fowleri ◽  
Reading Frame ◽  
Site Characteristic ◽  
High Conservation ◽  
Phosphofructokinase Activity ◽  
Cloned Cdna ◽  
Sequencing And Expression ◽  
Nægleria Fowleri

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.

scholarly journals Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

1991 ◽  
Vol 11 (2) ◽  
pp. 754-764 ◽  
Author(s):  
R Yano ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.

scholarly journals Identification, Cloning, and Expression of the CAMP-Like Factor Autotransporter Gene (cfa) of Bartonella henselae

2005 ◽  
Vol 73 (7) ◽  
pp. 4205-4213 ◽  
Author(s):  
Christine M. Litwin ◽  
Joel M. Johnson
Keyword(s):  
Amino Acid ◽  
Bartonella Henselae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
Cosmid Library ◽  
Group B Streptococci ◽  
Reading Frame ◽  
Calculated Molecular Weight ◽  
The Family

ABSTRACT The CAMP reaction was first described by Christie et al. (R. Christie, N. E. Atkins, and E. Munch-Petersen, Aust. J. Exp. Biol. 22:197-200, 1944) as the synergistic lysis of sheep red blood cells by Staphylococcus aureus sphingomyelinase and CAMP factor (cohemolysin), a secreted protein from group B streptococci. We observed a CAMP-like reaction when Bartonella henselae was grown in close proximity to S. aureus on 5% sheep blood agar. This study describes the cloning, sequencing, and characterization of a CAMP-like factor autotransporter gene (cfa) from B. henselae. A cosmid library of B. henselae ATCC 49793 was constructed using SuperCos1 in Escherichia coli XL1-Blue MR. Cosmids were screened for the CAMP reaction, and a quantitative cohemolysis microtiter assay was developed using purified sphingomyelinase. Cosmid clones with the strongest cohemolytic reaction had similar restriction enzyme patterns. A DNA fragment that expressed the cohemolysin determinant was subcloned in a 7,200-bp StuI-BamHI fragment which contained a 6,024-bp open reading frame. The deduced amino acid sequence showed homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane. They contain an N-terminal passenger region, the α-domain, and a C-terminal transporter region, the β-domain. The α-domain contained four, nearly identical 42-amino-acid repeats and showed homology to the family of RTX (repeat in toxin) hemolysins. The concentrated supernatant of the recombinant strain expressed a protein with a molecular mass of 180 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with the calculated molecular weight of the secreted α-domain. In conclusion, we have characterized a novel secreted cohemolysin autotransporter protein of B. henselae.

Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

2016 ◽  
Vol 56 (10) ◽  
pp. 1579 ◽  
Author(s):  
X. P. An ◽  
J. X. Hou ◽  
T. Y. Gao ◽  
B. Y. Cao
Keyword(s):  
Amino Acid ◽  
Litter Size ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Ovis Aries ◽  
Cloning And Expression ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Reading Frame ◽  
Kit Gene

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T > A and g.120466G > A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T > A mutation was a missense mutation (Tyr > Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T > A locus for three goat breeds (P < 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

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.

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