scholarly journals Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum

1996 ◽  
Vol 319 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Klaus WITTER ◽  
Dolores J CAHILL ◽  
Thomas WERNER ◽  
Irmgard ZIEGLER ◽  
Wolfgang RÖDL ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Single Gene ◽  
Pcr Amplification ◽  
Maximal Activity ◽  
Chromosomal Dna ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Reading Frame ◽  
E Coli ◽  
Km Value

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 °C. The Km value for GTP is 50 µM.

scholarly journals Production of Sepiapterin in Escherichia coli by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase

2002 ◽  
Vol 68 (6) ◽  
pp. 3138-3140 ◽  
Author(s):  
Hyun Joo Woo ◽  
Jee Yun Kang ◽  
Yong Kee Choi ◽  
Young Shik Park
Keyword(s):  
Escherichia Coli ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Aldose Reductase ◽  
High Performance ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
E Coli ◽  
Sepiapterin Reductase ◽  
Pcc 6803

ABSTRACT Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.

scholarly journals Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target

2019 ◽  
Vol 75 (9) ◽  
pp. 586-592
Author(s):  
Sonja Schüssler ◽  
Ilka Haase ◽  
Markus Perbandt ◽  
Boris Illarionov ◽  
Alexandra Siemens ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Active Sites ◽  
Putative Open Reading Frame ◽  
Molecular Replacement ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Reading Frame ◽  
Novel Drugs ◽  
Microbial Infections ◽  
Micromolar Range

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (K m = 53 µM; v max = 180 nmol mg−1 min−1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Å (R free = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D 5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

scholarly journals Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme

1994 ◽  
Vol 302 (1) ◽  
pp. 215-221 ◽  
Author(s):  
M Gütlich ◽  
E Jaeger ◽  
K P Rücknagel ◽  
T Werner ◽  
W Rödl ◽  
...  
Keyword(s):  
Maximum Activity ◽  
Open Reading Frames ◽  
Intermediate Step ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Rate Limiting Step ◽  
Enzyme Isoforms ◽  
Reading Frames

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

scholarly journals Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer

2002 ◽  
Vol 184 (7) ◽  
pp. 1932-1939 ◽  
Author(s):  
Karen C. Crasta ◽  
Kim-Lee Chua ◽  
Sumathi Subramaniam ◽  
Joachim Frey ◽  
Hilda Loh ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromosomal Dna ◽  
Pcr Cloning ◽  
Reading Frame ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Riemerella Anatipestifer ◽  
Hydrolysis Of

ABSTRACT Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam, from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp BamHI-BstXI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.

scholarly journals A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

1998 ◽  
Vol 180 (9) ◽  
pp. 2522-2530 ◽  
Author(s):  
Sergio L. Fuenmayor ◽  
Mark Wild ◽  
Alastair L. Boyes ◽  
Peter A. Williams
Keyword(s):  
Single Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Gas Chromatography Mass Spectrometry ◽  
Nucleotide Sequencing ◽  
Naphthalene Dioxygenase ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Selective Enrichment ◽  
E Coli

ABSTRACT Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kbBamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb(for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalenecis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAbwere two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designatednagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together inE. coli, nagG, nagH, andnagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts ofnagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene ordernagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.

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.

scholarly journals Characterisation of the GH gene cluster in a new-world monkey, the marmoset (Callithrix jacchus)

2002 ◽  
Vol 29 (1) ◽  
pp. 89-97 ◽  
Author(s):  
OC Wallis ◽  
M Wallis
Keyword(s):  
Gene Cluster ◽  
New World ◽  
World Monkey ◽  
Single Gene ◽  
Callithrix Jacchus ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
New World Monkey ◽  
Gh Gene ◽  
Close Relatives

In most mammals pituitary GH is encoded by a single gene with no close relatives. However, in man the GH gene has been shown to be one of a cluster of five closely related genes, four of which are expressed in the placenta. Rhesus monkey also expresses at least five closely related GH-like genes, although the genomic organisation of these has not been fully reported. Here we describe the cloning and characterisation of GH-like genes in a new-world monkey, the marmoset (Callithrix jacchus). This species possesses a cluster of eight GH-like 'genes'. The gene at the 5' end of this cluster encodes pituitary GH and is similar to that encoding human GH. Five of the eight marmoset 'genes' are probably pseudogenes, since they include mutations which would prevent normal expression, including stop codons and small insertions/deletions that would change the reading frame. In one case a large part of a gene is deleted, and in another a large insertion is introduced into an exon. The remaining two marmoset genes are potentially expressible, as proteins with sequences substantially different (at 25-30% of all residues) from that of marmoset GH itself; whether and in which tissue(s) such expression actually occurs is not yet known. None of the marmoset genes is clearly equivalent to any of the human GH-like genes expressed in the placenta, and this and phylogenetic analysis suggest that the duplications that gave rise to the marmoset GH gene cluster occurred independently of those that gave rise to the corresponding cluster in man. Although it includes more 'genes', the marmoset cluster extends over a shorter region of chromosomal DNA (about 35 kb) than does the human GH gene cluster (about 50 kb).

scholarly journals Direct Detection of eae-Positive Bacteria in Human and Veterinary Colorectal Specimens by PCR

1998 ◽  
Vol 36 (8) ◽  
pp. 2326-2330 ◽  
Author(s):  
A. L. Hubbard ◽  
D. J. Harrison ◽  
C. Moyes ◽  
S. McOrist
Keyword(s):  
Direct Detection ◽  
Pcr Amplification ◽  
Enteric Bacteria ◽  
Quick Method ◽  
Specific Sequence ◽  
Reading Frame ◽  
E Coli ◽  
Associated Lesions ◽  
Reference Strains ◽  
Pcr Test

A PCR test based on the amplification of aneae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenicEscherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli andC. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eaegenome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

1998 ◽  
Vol 64 (7) ◽  
pp. 2380-2385 ◽  
Author(s):  
Mónica Delgado ◽  
Héctor Toledo ◽  
Carlos A. Jerez
Keyword(s):  
Chromosomal Dna ◽  
Reading Frame ◽  
Leptospirillum Ferrooxidans ◽  
E Coli ◽  
Acid Protein ◽  
Chemotaxis Proteins ◽  
Acidophilic Bacterium ◽  
Sequencing And Expression ◽  
The Right

ABSTRACT We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacteriumLeptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillumchemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. ThelcrI gene showed both ς28-like and ς70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP fromEscherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed inE. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.

scholarly journals Control of 6-(d-threo-1′,2′-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity

1996 ◽  
Vol 314 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Markus GÜTLICH ◽  
Klaus WITTER ◽  
Julie BOURDAIS ◽  
Michel VERON ◽  
Wolfgang RÖDL ◽  
...  
Keyword(s):  
G Protein ◽  
Dictyostelium Discoideum ◽  
Time Course ◽  
De Novo ◽  
Mrna Level ◽  
Signalling Pathway ◽  
Cytosolic Fraction ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Specific Activities

6-(D-threo-1´,2´-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium discoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665–669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1´,2´,3´-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4–5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5´-[γ-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

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