Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes

1989 ◽  
Vol 9 (11) ◽  
pp. 5012-5021
Author(s):  
D Schlossman ◽  
D Withers ◽  
P Welsh ◽  
A Alexander ◽  
J Robertus ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Glutamic Acid Residue ◽  
Enzymatic Inactivation ◽  
Sequence Encoding ◽  
A Chain ◽  
Beta Galactosidase

The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.

scholarly journals Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

1989 ◽  
Vol 9 (11) ◽  
pp. 5012-5021 ◽  
Author(s):  
D Schlossman ◽  
D Withers ◽  
P Welsh ◽  
A Alexander ◽  
J Robertus ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Glutamic Acid Residue ◽  
Enzymatic Inactivation ◽  
Sequence Encoding ◽  
A Chain ◽  
Beta Galactosidase

The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.

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 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 Delivery of Ricin Toxin A-Chain by Peptide-Targeted Mesoporous Silica Nanoparticle-Supported Lipid Bilayers

2012 ◽  
Vol 1 (3) ◽  
pp. 348-353 ◽  
Author(s):  
Katharine Epler ◽  
David Padilla ◽  
Genevieve Phillips ◽  
Peter Crowder ◽  
Robert Castillo ◽  
...  
Keyword(s):  
Mesoporous Silica ◽  
Lipid Bilayers ◽  
Silica Nanoparticle ◽  
Supported Lipid Bilayers ◽  
Toxin A ◽  
Mesoporous Silica Nanoparticle ◽  
Ricin Toxin ◽  
A Chain

Detection of an abasic site in RNA with stem-loop DNA beacons: Application to an activity assay for Ricin Toxin A-Chain

2008 ◽  
Vol 70 (6) ◽  
pp. 945-953 ◽  
Author(s):  
Setu Roday ◽  
Matthew B. Sturm ◽  
Dukagjin M. Blakaj ◽  
Vern L. Schramm
Keyword(s):  
Abasic Site ◽  
Activity Assay ◽  
Toxin A ◽  
Stem Loop ◽  
Ricin Toxin ◽  
A Chain

Glutamic acid-141: a heme ‘bodyguard’ in anionic tobacco peroxidase

2007 ◽  
Vol 388 (4) ◽  
pp. 373-380 ◽  
Author(s):  
Dmitri M. Hushpulian ◽  
Andrew A. Poloznikov ◽  
Pavel A. Savitski ◽  
Alexandra M. Rozhkova ◽  
Tatyana A. Chubar ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Active Center ◽  
Structural Alignment ◽  
Wild Type ◽  
E Coli ◽  
Anionic Peroxidase ◽  
Glutamic Acid Residue ◽  
Plant Peroxidases ◽  
Tobacco Peroxidase

Abstract The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it ‘guards’ heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.

Sign in / Sign up

Export Citation Format

Share Document