Genetic Manipulation of Key Photosynthetic Enzymes in the C4 Plant Flaveria bidentis

1997 ◽  
Vol 24 (4) ◽  
pp. 477 ◽  
Author(s):  
Robert T. Furbank ◽  
Julie A. Chitty ◽  
Colin L.D. Jenkins ◽  
William C. Taylor ◽  
Stephen J. Trevanion ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
Genetic Manipulation ◽  
Photosynthetic Performance ◽  
Covalent Modification ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Enzymes ◽  
Flaveria Bidentis

The NADP-malic enzyme type C4 dicot Flaveria bidentis (L.) Kuntze was transformed with antisense and cosense gene constructs that resulted in specific decreases in single photosynthetic enzymes. The enzymes targeted were ribulose-1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39] (Rubisco), pyruvate, Pi dikinase [EC 2.7.9.1] (PPDK) and NADP malate dehydrogenase [EC 1.1.1.82] (NADP-MDH). These enzymes were chosen as targets because they have low activity compared to photosynthetic rates (Rubisco), are subject to complex covalent regulation (NADP-MDH), or both (PPDK). T1 progeny of a number of lines of these transformants were examined for the effects of these gene constructs on enzyme levels and photosynthetic performance. Rubisco antisense transformants expressing between 15 and 100% of wild-type enzyme activity were obtained. Pyruvate, Pi dikinase antisense lines were obtained with 40–100% wild-type levels. NADP malate dehydrogenase sense constructs caused a co-suppression of enzyme activity with some lines containing less than 2% of wild- type activity. Under saturating illumination, the control coefficients for these enzymes were determined to be up to 0.7 for Rubisco, 0.2–0.3 for PPDK and effectively zero for NADP-MDH. The implications of these observations for the regulation of photosynthetic flux and metabolism in C4 plants and the role of regulation by covalent modification are discussed.

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes

1990 ◽  
Vol 10 (12) ◽  
pp. 6257-6263
Author(s):  
A Frankel ◽  
P Welsh ◽  
J Richardson ◽  
J D Robertus
Keyword(s):  
Enzyme Activity ◽  
Glutamic Acid ◽  
Structural Information ◽  
Wild Type ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Enzymatic Inactivation ◽  
A Chain

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals The plasma membrane Ca2+ pump mutant lysine591 → arginine retains some activity, but is still inactivated by fluorescein isothiocyanate

1996 ◽  
Vol 317 (1) ◽  
pp. 41-44 ◽  
Author(s):  
Hugo P. ADAMO ◽  
Adelaida G. FILOTEO ◽  
John T. PENNISTON
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Plasma ◽  
Fluorescein Isothiocyanate ◽  
Covalent Modification ◽  
Transport Activity ◽  
Wild Type ◽  
Pump Function ◽  
Irreversible Inhibition ◽  
Lysine Residues

Inactivation of the wild-type human plasma membrane Ca2+ pump (isoform 4b) by fluorescein isothiocyanate is accompanied by covalent modification of Lys591. The mutation of Lys591 to arginine reduced the Ca2+ transport activity to 35% of the wild-type, and diminished the amount of acylphosphate formed from ATP by a corresponding amount. When this mutant was treated with fluorescein isothiocyanate, the enzyme was still irreversibly inactivated, even though no reactive residue was available at position 591. The results show that, although Ca2+ pump function is sensitive to the residue at position 591, Lys591 is not essential for enzyme activity. They also demonstrate that irreversible inhibition of the plasma membrane Ca2+ pump by fluorescein isothiocyanate does not require the covalent modification of Lys591. This indicates that fluorescein isothiocyanate reacts with lysine residues at other positions in addition to Lys591.

scholarly journals Differential CYP 2D6 Metabolism Alters Primaquine Pharmacokinetics

2015 ◽  
Vol 59 (4) ◽  
pp. 2380-2387 ◽  
Author(s):  
Brittney M. J. Potter ◽  
Lisa H. Xie ◽  
Chau Vuong ◽  
Jing Zhang ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Antimalarial Activity ◽  
Wild Type ◽  
Phenolic Metabolites ◽  
Species Specific ◽  
Human And Mouse ◽  
Different Levels ◽  
Cyp 2D6

ABSTRACTPrimaquine (PQ) metabolism by the cytochrome P450 (CYP) 2D family of enzymes is required for antimalarial activity in both humans (2D6) and mice (2D). Human CYP 2D6 is highly polymorphic, and decreased CYP 2D6 enzyme activity has been linked to decreased PQ antimalarial activity. Despite the importance of CYP 2D metabolism in PQ efficacy, the exact role that these enzymes play in PQ metabolism and pharmacokinetics has not been extensively studiedin vivo. In this study, a series of PQ pharmacokinetic experiments were conducted in mice with differential CYP 2D metabolism characteristics, including wild-type (WT), CYP 2D knockout (KO), and humanized CYP 2D6 (KO/knock-in [KO/KI]) mice. Plasma and liver pharmacokinetic profiles from a single PQ dose (20 mg/kg of body weight) differed significantly among the strains for PQ and carboxy-PQ. Additionally, due to the suspected role of phenolic metabolites in PQ efficacy, these were probed using reference standards. Levels of phenolic metabolites were highest in mice capable of metabolizing CYP 2D6 substrates (WT and KO/KI 2D6 mice). PQ phenolic metabolites were present in different quantities in the two strains, illustrating species-specific differences in PQ metabolism between the human and mouse enzymes. Taking the data together, this report furthers understanding of PQ pharmacokinetics in the context of differential CYP 2D metabolism and has important implications for PQ administration in humans with different levels of CYP 2D6 enzyme activity.

Antisense RNA inhibition of pyruvate, orthophosphate dikinase and NADP malate dehydrogenase in the C4 plant Flaveria bidentis: analysis of plants with a mosaic phenotype

1999 ◽  
Vol 26 (6) ◽  
pp. 537 ◽  
Author(s):  
Anthony R. Ashton ◽  
Robert T. Furbank ◽  
Stephen J. Trevanion
Keyword(s):  
Transgenic Plants ◽  
Malate Dehydrogenase ◽  
Antisense Rna ◽  
Wild Type ◽  
Pyruvate Orthophosphate Dikinase ◽  
Orthophosphate Dikinase ◽  
Transgenic Lines ◽  
Rna Inhibition ◽  
Target Enzyme ◽  
Flaveria Bidentis

Antisense RNA suppression of either pyruvate, orthophosphate dikinase [EC 2.7.9.1] or NADP malate dehydrogenase [EC 1.1.1.82] gene expression in the C4 dicot Flaveria bidentis L. var. Kuntze produced several independent transgenic lines with leaves showing heritable, mosaic phenotypes. The appearance of these plants was highly variable, with leaves that were either predominantly green, predominantly yellow, or a mixture of the two. The yellow sectors appeared to be clonal in origin. For both sets of transgenic plants, the green and yellow sectors showed a reduction in the activity of the respective target enzyme compared to wild-type leaves. The mRNA of the target enzyme was reduced in both green and yellow sectors of leaves of both types of transformants compared to leaves from wild-type plants. The yellow sectors had decreased amounts of other photosynthetic enzymes on an area basis, but most enzyme activities and electron transport rates were similar to the green sectors on a chlorophyll basis. The mosaic phenotype could not be attributed simply to the degree of suppression of the target enzyme, because we have also obtained uniformly green plants with similar or greater enzyme suppression. The importance of this spatial variability in the effectiveness of the antisense transgenes for the analysis of transgenic plants in general is discussed.

scholarly journals Role of a Bacterial Organic Hydroperoxide Detoxification System in Preventing Catalase Inactivation

2004 ◽  
Vol 279 (50) ◽  
pp. 51908-51914 ◽  
Author(s):  
Ge Wang ◽  
Richard C. Conover ◽  
Stephane Benoit ◽  
Adriana A. Olczak ◽  
Jonathan W. Olson ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Stress Resistance ◽  
Molar Ratio ◽  
Wild Type ◽  
Organic Hydroperoxide ◽  
Organic Hydroperoxides ◽  
Detoxification System ◽  
H Pylori ◽  
Mutant Cells

In the gastric pathogenHelicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase inahpCmutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides onH. pyloricatalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase.H. pyloricatalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in theahpCmutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found inahpCmutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in theahpCmutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.

scholarly journals Male courtship song drives escape responses that are suppressed for successful mating

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eliane Arez ◽  
Cecilia Mezzera ◽  
Ricardo M. Neto-Silva ◽  
Márcia M. Aranha ◽  
Sophie Dias ◽  
...  
Keyword(s):  
Escape Response ◽  
Walking Speed ◽  
Genetic Manipulation ◽  
Courtship Song ◽  
Male Courtship ◽  
Wild Type ◽  
Crucial Component ◽  
Escape Responses ◽  
Sexually Mature

AbstractPersuasion is a crucial component of the courtship ritual needed to overcome contact aversion. In fruit flies, it is well established that the male courtship song prompts receptivity in female flies, in part by causing sexually mature females to slow down and pause, allowing copulation. Whether the above receptivity behaviours require the suppression of contact avoidance or escape remains unknown. Here we show, through genetic manipulation of neurons we identified as required for female receptivity, that male song induces avoidance/escape responses that are suppressed in wild type flies. First, we show that silencing 70A09 neurons leads to an increase in escape, as females increase their walking speed during courtship together with an increase in jumping and a reduction in pausing. The increase in escape response is specific to courtship, as escape to a looming threat is not intensified. Activation of 70A09 neurons leads to pausing, confirming the role of these neurons in escape modulation. Finally, we show that the escape displays by the female result from the presence of a courting male and more specifically from the song produced by a courting male. Our results suggest that courtship song has a dual role, promoting both escape and pause in females and that escape is suppressed by the activity of 70A09 neurons, allowing mating to occur.

scholarly journals Genetic control of metabolism: enzyme studies of the obese and adipose mutants in the mouse

1972 ◽  
Vol 20 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Grahame Bulfield
Keyword(s):  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
Primary Lesion ◽  
Gas Liquid Chromatography ◽  
Wild Type ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Lyase Activity ◽  
Metabolism Enzyme ◽  
Pheno Type

SUMMARYThe activity of several enzymes has been determined in the livers of homozygous obese and adipose mice, their normal litter-mates, and phenocopies induced in normal mice by aurothioglucose (ATG) injections.Obese, adipose and ATG mice had higher activities of ATP citrate lyase, malic enzyme (NADP malate dehydrogenase) and pyruvate kinase than normal mice. Heterozygote activities are indistinguishable from wild-type. There was no difference between normal and fat litter-mates in the activity of malate dehydrogenase (NAD-linked), lactate dehydrogenase, isocitrate dehydrogenase and fumarase.Crosses between mice doubly heterozygous for roth the ad and ob genes produced offspring that were only ‘fat’ or ‘normal’ and no offspring could be phenotypically recognized as the double mutant, either physically or in terms of ATP citrate lyase activity.Gas–liquid chromatography of the fatty acids of the depot fat showed no differences between any of the types of litter-mate.The alterations found in enzyme activity in obese and adipose mice are compared to several other enzyme activity differences reported in the literature for obese mice. These are discussed in relation to genetical criteria that may be estarlished to assess, from quantitative data, whether an enzyme is the site of the primary lesion in a mutant pheno-type. Some general observations are made on genetics and the control of metabolism.

scholarly journals Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation

2003 ◽  
Vol 185 (5) ◽  
pp. 1509-1517 ◽  
Author(s):  
Yehouda Marcus ◽  
Hagit Altman-Gueta ◽  
Aliza Finkler ◽  
Michael Gurevitz
Keyword(s):  
Redox Regulation ◽  
Inhibitory Effect ◽  
Cysteine Residues ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Enzyme Form ◽  
Oxygenase Activity ◽  
In The Wild ◽  
Cross Links

ABSTRACT Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.

scholarly journals Salmonella enterica serovar Typhimurium sseK3 induces apoptosis and enhances glycolysis in macrophages

2020 ◽  
Author(s):  
Chuan Yu ◽  
Fuyu Du ◽  
Chunjie Zhang ◽  
Yinju Li ◽  
Chengshui Liao ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Salmonella Enterica ◽  
Caspase 3 ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Caspase 8 ◽  
Wild Type ◽  
Caspase 9 ◽  
Serovar Typhimurium ◽  
The Relationship

Abstract Background: Salmonella enterica serovar Typhimurium ( S. Typhimurium) is an important infectious disease pathogen that can survive and replicate in macrophages. Glycolysis is essential for immune responses against S. Typhimurium infection in macrophages, and is also associated with apoptosis. S. Typhimurium secreted effector K3 (SseK3) was recently identified as a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis in cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in S. Typhimurium-infected macrophages. Results: Macrophages were infected with S. Typhimurium SL1344 wild-type (WT), Δ sseK3 mutant or sseK3 -complemented strain, and the effects of sseK3 on apoptosis and glycolysis were determined. The adherence and invasion in the Δ sseK3 mutant group were similar to that in the WT and sseK3 -complemented groups, indicating that SseK3 was not essential for the adherence and invasion of S. Typhimurium in macrophages. However, the percentage of apoptosis in the Δ sseK3 mutant group was much lower than that in the WT and sseK3 -complemented groups. Caspase-3, caspase-8, and caspase-9 enzyme activity in the Δ sseK3 mutant group were significantly lower than in the WT group and sseK3 -complemented groups, indicating that sseK3 could improve the caspase-3, caspase-8, and caspase-9 enzyme activity. We also found that there were no significant differences in pyruvic acid levels between the three groups, but the lactic acid level in the Δ sseK3 mutant group was much lower than that in the WT and sseK3 -complemented groups. The ATP levels in the Δ sseK3 mutant group were remarkably higher than those in the WT and sseK3 -complemented groups. These indicated that the sseK3 enhanced the level of glycolysis in macrophages infected by S. Typhimurium. Conclusions: S. Typhimurium sseK3 is likely involved in promoting macrophage apoptosis and modulating glycolysis in macrophages. Our results could improve our understanding of the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3 .

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