scholarly journals Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening

2020 ◽  
Vol 11 (24) ◽  
pp. 6134-6148 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
De Novo ◽  
Library Screening ◽  
Natural Reaction

De novo enzymes capable of efficiently catalysis of a non-natural reaction are obtained through minimalist design plus computationally-focused variant library screening.

scholarly journals Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo

1975 ◽  
Vol 146 (1) ◽  
pp. 121-126 ◽  
Author(s):  
E G Fragoulis ◽  
C E Sekeris
Keyword(s):  
Enzyme Activity ◽  
Steroid Hormones ◽  
De Novo ◽  
Steroid Hormone ◽  
Dopa Decarboxylase ◽  
Double Labelling ◽  
Labelling Technique ◽  
Immune Precipitation ◽  
Enzyme Molecules ◽  
Stimulation Of

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.

scholarly journals Acid phosphatase and cathepsin activity in cuttlefish (Sepia officinalis) eggs: the effects of Ag, Cd, and Cu exposure

2010 ◽  
Vol 67 (7) ◽  
pp. 1517-1523 ◽  
Author(s):  
Thomas Lacoue-Labarthe ◽  
Estelle Le Bihan ◽  
David Borg ◽  
Noussithé Koueta ◽  
Paco Bustamante
Keyword(s):  
Heavy Metals ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Embryonic Development ◽  
Enzyme Activities ◽  
De Novo ◽  
Sepia Officinalis ◽  
Activity Levels ◽  
Cathepsin Activity ◽  
Hatchling Weight

Abstract Lacoue-Labarthe, T., Le Bihan, E., Borg, D., Koueta, N., and Bustamante, P. 2010. Acid phosphatase and cathepsin activity in cuttlefish (Sepia officinalis) eggs: the effects of Ag, Cd, and Cu exposure. – ICES Journal of Marine Science, 67: 1517–1523. Changes in the activity levels of acid phosphatase (AcP) and cathepsin during cuttlefish embryo development are described, as are the effects of exposure to heavy metals. Enzyme activity kinetics appear to be linked to the developmental stage. The activities of both enzymes increased during the final days of development, suggesting de novo production by the maturing embryo in the digestive gland. The effects of selected heavy metals, Ag (0.06, 1.2, 60, 1200 ng l−1), Cd (31, 61, 305, 610 ng l−1), and Cu (0.23, 2.3, 23, 230 µg l−1), were assessed based on AcP and cathepsin activities at the end of embryonic development and on hatchling weight. Enzyme activities were not impacted by Ag but were significantly inhibited by Cd, at all four concentrations for AcP and at 610 ng l−1 for cathepsin. Cu (at 2.3 µg l−1) stimulated AcP activity. No cause–effect relationship was found between the effects of metals on the enzyme activities and hatchling weight, suggesting that heavy metals could affect other physiological functions during embryogenesis.

scholarly journals De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

1996 ◽  
Vol 16 (8) ◽  
pp. 4555-4565 ◽  
Author(s):  
P M Vertino ◽  
R W Yen ◽  
J Gao ◽  
S B Baylin
Keyword(s):  
Enzyme Activity ◽  
De Novo ◽  
Cpg Island ◽  
Human Fibroblasts ◽  
Methylation Status ◽  
Cpg Islands ◽  
Parental Cell Line ◽  
Direct Role ◽  
Cpg Island Methylation ◽  
De Novo Methylation

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

scholarly journals Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 996 ◽  
Author(s):  
Ohm Prakash ◽  
Jana Führing ◽  
John Post ◽  
Sharon Shepherd ◽  
Thomas Eadsforth ◽  
...  
Keyword(s):  
Leishmania Major ◽  
De Novo ◽  
Protozoan Parasite ◽  
Library Screening ◽  
Uridine Diphosphate ◽  
Fragment Library ◽  
Antileishmanial Drug ◽  
Small Set ◽  
Diphosphate Glucose ◽  
Nucleotide Sugars

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

Thymidylate kinase from Acetabularia. II. Regulation during the life cycle

1983 ◽  
Vol 64 (1) ◽  
pp. 27-36
Author(s):  
E.J. de Groot ◽  
H.G. Schweiger
Keyword(s):  
Enzyme Activity ◽  
Life Cycle ◽  
Kinase Activity ◽  
De Novo ◽  
Nuclear Genome ◽  
De Novo Synthesis ◽  
Cell Extracts ◽  
Phase Regulation ◽  
Generative Phase ◽  
Low Activity

The activity of dTMP kinase (EC 2.7.4.9) was estimated during the development of Acetabularia. Enzyme activity was increased at the beginning of the generative phase. Regulation of the dTMP kinase activity was observed even in the absence of the nucleus. More than 30 days after enucleation the enzyme activity increased two- to threefold. The increase in activity was inhibited by puromycin and cycloheximide but not by rifampicin and chloramphenicol. These results indicate that the enzyme is coded by the nuclear genome and translated on 80 S ribosomes. From mixing experiments with low-activity and high-activity cell extracts it is concluded that the regulation is due to de novo synthesis of the enzyme.

Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

1983 ◽  
Vol 29 (6) ◽  
pp. 681-688 ◽  
Author(s):  
Daniel J. Nieto ◽  
Robin A. Woods
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Ultraviolet Light ◽  
Temperature Sensitivity ◽  
De Novo ◽  
Feedback Inhibition ◽  
Temperature Sensitive ◽  
Ethylmethane Sulphonate ◽  
Interallelic Complementation ◽  
Different Levels

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.

scholarly journals Transcription and redox enzyme activities: comparison of equilibrium and disequilibrium levels in the three-spined stickleback

2013 ◽  
Vol 280 (1755) ◽  
pp. 20122974 ◽  
Author(s):  
M. Nikinmaa ◽  
R. J. S. McCairns ◽  
M. W. Nikinmaa ◽  
K. A. Vuori ◽  
M. Kanerva ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
High Throughput ◽  
Population Differentiation ◽  
Gasterosteus Aculeatus ◽  
De Novo ◽  
Time Lag ◽  
Redox Enzyme ◽  
Activity Data ◽  
Acute Changes

Evolutionary and acclimatory responses require functional variability, but in contrast with mRNA and protein abundance data, most physiological measurements cannot be obtained in a high-throughput manner. Consequently, one must either rely on high-throughput transcriptomic or proteomic data with only predicted functional information, or accept the limitation that most physiological measurements can give fewer data than those provided by transcriptomics or proteomics. We evaluated how transcriptional and redox enzyme activity data agreed with regard to population differentiation (i.e. a system in steady state in which any time lag between transcription, translation and post-translational effects would be irrelevant) and in response to an acute 6°C increase in temperature (i.e. a disequilibrium state wherein translation could not have caught up with transcription) in the three-spined stickleback ( Gasterosteus aculeatus ). Transcriptional and enzyme activity data corresponded well with regard to population differentiation, but less so with regard to acute temperature increase. The data thus suggest that transcriptional and functional measurements can lead to similar conclusions when a biological system is in a steady state. The responses to acute changes must, as has been demonstrated earlier, be based on changes in cellular conditions or properties of existing proteins without significant de novo synthesis of new gene products.

scholarly journals Rates of degradation and synthesis of glycosidases de novo during growth and differentiation of Dictyostelium discoideum

1975 ◽  
Vol 148 (2) ◽  
pp. 169-177 ◽  
Author(s):  
D Every ◽  
J M Ashworth
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Life Cycle ◽  
Dictyostelium Discoideum ◽  
Growth Medium ◽  
Growth Phase ◽  
Enzyme Activities ◽  
De Novo ◽  
Antibody Preparation ◽  
Differentiated Cells

1. Injection of a purified preparation of β-N-acetylglucosaminidase from the spent growth medium of myxamoebae of Dictyostelium discoideum into rabbits gave rise to an antibody preparation containing both anti-α-glucosidase and anti-β-acetylglucosaminidase activities. 2. These two activities were shown to reside in different immunoglobulin molecules and it was concluded that the β-N-acetylglucosaminidase preparation contained trace amounts of highly antigenic α-glucosidase. 3. A single precipitin band having β-N-acetylglucosaminidase activity was formed in Ouchterlony plates when this antibody preparation was tested against extracts obtained from differentiated cells or from myxamoebae grown either axenically or on bacteria. 4. The antibody preparation was used to show that both β-N-acetylglucosaminidase and α-glucosidase molecules are synthesized de novo from isotopically labelled amino acids during both the growth and differentiation phases of the life cycle and to show that neither of these proteins is significantly degraded during the growth phase or during the first 9h of differentiation. 5. The rates of accumulation of these assayable enzyme activities are thus equal to their rates of synthesis during growth and early differentiation. 6. The factors regulating cellular enzyme activity during the life cycle of D. discoideum are discussed.

scholarly journals Identification of dihydropteridine reductase in human platelets

Blood ◽  
1979 ◽  
Vol 53 (1) ◽  
pp. 116-121
Author(s):  
HT Abelson ◽  
C Gorka ◽  
GP Beardsley
Keyword(s):  
Enzyme Activity ◽  
Biogenic Amine ◽  
De Novo ◽  
Human Platelets ◽  
Partial Purification ◽  
Dihydropteridine Reductase ◽  
Transport Function ◽  
Reductase Deficiency ◽  
Physical Data ◽  
Normal Human

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.

Use of Immunotitration to Demonstrate Phytochrome-Mediated Synthesis de novo of Chalcone Synthase and Phenylalanine Ammonia Lyase in Mustard Seedling Cotyledons

1986 ◽  
Vol 41 (1-2) ◽  
pp. 61-68 ◽  
Author(s):  
Reiner Brödenfeldt ◽  
Hans Mohr
Keyword(s):  
Enzyme Activity ◽  
Phenylalanine Ammonia Lyase ◽  
Chalcone Synthase ◽  
De Novo ◽  
Ribulose Bisphosphate Carboxylase ◽  
Enzyme Protein ◽  
Experimental Conditions ◽  
Bisphosphate Carboxylase ◽  
Antigenic Material ◽  
Induction Of Enzymes

Abstract The suggestion [H. J. Newbury and H. Smith, Eur. J. Biochem. 117, 575-580 (1981)] of a posttranslational activation of accumulated enzyme precursor by phytochrome was tested in the case of phytochrome-mediated induction of chalcone synthase (EC 2.3.1.74) in mustard seedlings. The approach of immunotitration of enzyme activity was adopted since it allows to relate the amount of antigenic material present in a particular extract to the extract’s enzymatic activity. The data obtained show that under all experimental conditions enzyme activity of an extract is proportional to the amount of im munoresponsive material. No indication was found either of lightdependent modification of the enzyme or of enzymatically inactive immunoresponsive material in extracts prepared from dark-grown mustard seedlings or from light-treated seedlings placed again in darkness. In model experiments with purified ribulose-bisphosphate carboxylase (EC 4.1.1.39) heat-inactivated enzyme was detected by the antibodies. Experiments on induction of phenylalanine ammonia lyase (EC 4.3.1.5) by phytochrome in mustard cotyledons support the result - synthesis de novo of enzyme protein - obtained previously [W. Tong and P. Schöpfer, Proc. Nat. Acad. Sci. U.S.A. 43, 4017-4021 (1976)] with the technique of density labelling. It is concluded that the phytochrome-mediated induction of enzymes involved in anthocyanin formation in mustard cotyledons is due to synthesis de novo of enzyme protein.

Sign in / Sign up

Export Citation Format

Share Document