scholarly journals IMP Dehydrogenase from the Protozoan Parasite Toxoplasma gondii

2005 ◽  
Vol 49 (6) ◽  
pp. 2172-2179 ◽  
Author(s):  
William J. Sullivan ◽  
Stacy E. Dixon ◽  
Catherine Li ◽  
Boris Striepen ◽  
Sherry F. Queener
Keyword(s):  
Toxoplasma Gondii ◽  
Mycophenolic Acid ◽  
De Novo ◽  
Protozoan Parasite ◽  
Gene Encoding ◽  
Imp Dehydrogenase ◽  
Reading Frame ◽  
Folate Pathway ◽  
Acid Protein ◽  
Derivatives Of

ABSTRACT The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii ) of 0.03 ± 0.004 μM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable β-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.

scholarly journals Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis

2009 ◽  
Vol 75 (18) ◽  
pp. 5938-5942 ◽  
Author(s):  
Yumei Li ◽  
Lili Lu ◽  
Hongmei Wang ◽  
Xiaodong Xu ◽  
Min Xiao
Keyword(s):  
Cell Surface ◽  
Surface Engineering ◽  
Yeast Cells ◽  
Penicillium Expansum ◽  
Yeast Nitrogen Base ◽  
Gene Encoding ◽  
Nitrogen Base ◽  
Reading Frame ◽  
Acid Protein ◽  
Casamino Acids

ABSTRACT A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.

scholarly journals Identification of an Alternative Nucleoside Triphosphate: 5′-Deoxyadenosylcobinamide Phosphate Nucleotidyltransferase in Methanobacterium thermoautotrophicum ΔH

2000 ◽  
Vol 182 (15) ◽  
pp. 4227-4233 ◽  
Author(s):  
Michael G. Thomas ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Kinase Activity ◽  
De Novo ◽  
Methanobacterium Thermoautotrophicum ◽  
Cell Extract ◽  
Nucleoside Triphosphate ◽  
De Novo Synthesis ◽  
Anaerobic Conditions ◽  
Gene Encoding ◽  
Reading Frame ◽  
Archaeal Gene

ABSTRACT Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5′-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domainArchaea. The corresponding ORF open reading frame fromMethanobacterium thermoautotrophicum ΔH (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum ΔH cell extract, suggesting that this organism may not have this activity. ThecobY gene complemented a cobU mutant ofS. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis.cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.

scholarly journals Cloning of a Mucin-Desulfating Sulfatase Gene fromPrevotella Strain RS2 and Its Expression Using aBacteroides Recombinant System

2000 ◽  
Vol 182 (11) ◽  
pp. 3002-3007 ◽  
Author(s):  
Damian P. Wright ◽  
Catriona G. Knight ◽  
Shanthi G. Parkar ◽  
David L. Christie ◽  
Anthony M. Roberton
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Signal Sequence ◽  
Active Form ◽  
Open Reading Frame ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

ABSTRACT A gene encoding the mucin-desulfating sulfatase inPrevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into aBacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

scholarly journals Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes

1992 ◽  
Vol 287 (3) ◽  
pp. 785-790 ◽  
Author(s):  
J Balzarini ◽  
E De Clercq
Keyword(s):  
Mycophenolic Acid ◽  
Marked Effect ◽  
De Novo ◽  
Potent Inhibitor ◽  
Assay Method ◽  
The Novel ◽  
Inhibitory Effects ◽  
Intact Cells ◽  
Imp Dehydrogenase

A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells.

scholarly journals The PET54 gene of Saccharomyces cerevisiae: characterization of a nuclear gene encoding a mitochondrial translational activator and subcellular localization of its product.

Genetics ◽  
1989 ◽  
Vol 122 (2) ◽  
pp. 297-305 ◽  
Author(s):  
M C Costanzo ◽  
E C Seaver ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Reading Frame ◽  
Acid Protein ◽  
Translational Activator ◽  
The Right ◽  
A Minor

Abstract The product of the nuclear Saccharomyces cerevisiae gene PET54 is specifically required, along with at least two other nuclear gene products, for translation of the mitochondrial mRNA encoding subunit III of cytochrome c oxidase (coxIII). We have genetically mapped PET54 (to the right arm of chromosome VII, 4.8 cM centromere-distal to SUF15), and have biochemically characterized the gene and its product. We determined the nucleotide sequence of a 1.6-kb DNA fragment carrying PET54 and identified the PET54 reading frame by determining the sequence of an ochre mutant allele as well as frameshift and frameshift-revertant alleles of the gene. The wild-type PET54 gene encodes a slightly basic 293-amino acid protein. PET54 is expressed from two mRNAs, both with unusual features: a major transcript with an extremely short 5'-untranslated leader, and a minor transcript with a relatively long 5'-leader carrying three short open reading frames. Antiserum raised against a trpE-PET54 fusion protein was used to probe subcellular fractions. These experiments showed that the PET54 protein is specifically associated with mitochondria, suggesting that it is likely to act directly in coxIII translation.

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.

Pharmacodynamic assessment of mycophenolic acid-induced immunosuppression by measuring IMP dehydrogenase activity

1995 ◽  
Vol 41 (2) ◽  
pp. 295-299 ◽  
Author(s):  
L J Langman ◽  
D F LeGatt ◽  
R W Yatscoff
Keyword(s):  
Enzyme Activity ◽  
Mycophenolic Acid ◽  
Whole Blood ◽  
De Novo ◽  
Immunosuppressive Drugs ◽  
Immunosuppressive Drug ◽  
Therapeutic Drug ◽  
Imp Dehydrogenase ◽  
Impdh Activity ◽  
Key Enzyme

Abstract Pharmacodynamic monitoring of the biological effect of immunosuppressive drugs provides an alternative to traditional therapeutic drug monitoring. We chose this method to investigate mycophenolic acid (MPA), an immunosuppressive drug that mediates its effect by inhibition of IMP dehydrogenase (IMPDH), a key enzyme in the de novo biosynthesis of purines. Using an assay developed for measuring IMPDH activity in whole blood, we found the concentration of MPA required for 50% inhibition of enzyme activity to be in the range of 2.0-5.0 mg/L for both human and rabbit blood. The amount of enzyme activity in whole blood depended on the concentration of the leukocytes, was unaffected by the type of anticoagulant used, and was stable in blood specimens stored for as long as 48 h at 4 degrees C. An inverse relationship was found between plasma MPA concentrations and IMPDH activity in rabbits administered a single dose of RS-61443, the prodrug of MPA. Maximal inhibition of IMPDH activity (by approximately 60%) occurs at peak concentrations of MPA; as the concentration of the drug decreases postdose, the enzyme activity gradually increases with little or no inhibition being observed 24 h postdose.

scholarly journals Single-Copy IMH3 Allele Is Sufficient To Confer Resistance to Mycophenolic Acid in Candida albicans and To Mediate Transformation of Clinical Candida Species

2001 ◽  
Vol 69 (1) ◽  
pp. 108-114 ◽  
Author(s):  
Janna Beckerman ◽  
Hiroji Chibana ◽  
Joshua Turner ◽  
P. T. Magee
Keyword(s):  
Candida Albicans ◽  
Mycophenolic Acid ◽  
De Novo ◽  
Selectable Marker ◽  
Single Copy ◽  
Clinical Isolates ◽  
Wild Type ◽  
Reading Frame ◽  
Sequence Elements ◽  
Dominant Selectable Marker

ABSTRACT Parasexual genetic analysis of Candida albicansutilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced theIMH3 r gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213–218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331–2338, 1997) have shown that the wild-typeIMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that theIMH3 r gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis. We also identified the presence of autonously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a φ-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3 r allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

scholarly journals Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

2018 ◽  
Vol 19 (7) ◽  
pp. 2111 ◽  
Author(s):  
Elena Maresi ◽  
Giacomo Janson ◽  
Silvia Fruncillo ◽  
Alessandro Paiardini ◽  
Rosario Vallone ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Active Sites ◽  
De Novo ◽  
Mutational Analysis ◽  
Homology Modelling ◽  
Functional Characterization ◽  
Protozoan Parasite ◽  
Cysteine Synthase ◽  
Transsulfuration Pathway ◽  
Reaction Specificity

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,β- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.

scholarly journals Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes

1990 ◽  
Vol 272 (2) ◽  
pp. 369-376 ◽  
Author(s):  
L E Kellett ◽  
D M Poole ◽  
L M Ferreira ◽  
A J Durrant ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Pseudomonas Fluorescens ◽  
Crystalline Cellulose ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Binding Domains ◽  
Cellulose Binding ◽  
Derivatives Of

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5′ end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

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