scholarly journals Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe–4S] cluster with one non-cysteine ligand

1989 ◽  
Vol 264 (1) ◽  
pp. 275-284 ◽  
Author(s):  
S J George ◽  
F A Armstrong ◽  
E C Hatchikian ◽  
A J Thomson
Keyword(s):  
Amino Acid ◽  
Pyrolytic Graphite ◽  
Direct Electrochemistry ◽  
Spectroscopic Characterization ◽  
Cysteine Residue ◽  
Side Chain ◽  
Iron Protein ◽  
Plausible Model ◽  
The One

Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one [3Fe-4S]1+,0 and one [4Fe-4S]2+,1+ core cluster when aerobically isolated. The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters. Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-‘edge’ electrode promoted by neomycin shows that, when reduced, the [3Fe-4S]0 centre reacts rapidly with Fe(II) ion to form a [4Fe-4S]2+ cluster. The latter, which can be reduced at a redox potential similar to that of the other [4Fe-4S] cluster, must include non-thiolate ligation. We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin. The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d. and e.p.r. spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state. This cluster provides a plausible model for the ligation states of the [4Fe-4S]1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase.

scholarly journals Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

2001 ◽  
Vol 67 (4) ◽  
pp. 1601-1606 ◽  
Author(s):  
Mitsunori Ishiguro ◽  
Satoshi Kaneko ◽  
Atsushi Kuno ◽  
Yoshinori Koyama ◽  
Shigeki Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Catalytic Function ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

scholarly journals Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus

1989 ◽  
Vol 264 (1) ◽  
pp. 265-273 ◽  
Author(s):  
F A Armstrong ◽  
S J George ◽  
R Cammack ◽  
E C Hatchikian ◽  
A J Thomson
Keyword(s):  
Low Temperature ◽  
Pyrolytic Graphite ◽  
Spectroscopic Characterization ◽  
Carbon Electrode ◽  
Standard Hydrogen Electrode ◽  
Hydrogen Electrode ◽  
Iron Cluster ◽  
Desulfovibrio Gigas ◽  
Acid Labile

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-‘edge’ carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0′ = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.

scholarly journals Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2

2003 ◽  
Vol 376 (1) ◽  
pp. 277-283 ◽  
Author(s):  
Mauricio BAEZ ◽  
Patricio H. RODRÍGUEZ ◽  
Jorge BABUL ◽  
Victoria GUIXÉ
Keyword(s):  
Escherichia Coli ◽  
Cysteine Residue ◽  
Active Species ◽  
Native Enzyme ◽  
Functional Roles ◽  
Allosteric Effector ◽  
Rapid Inactivation ◽  
Modified Enzyme ◽  
The One

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme. The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme. The two modified residues were identified as Cys-238 and Cys-295. In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue. Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants (Km, kcat) being almost identical to the ones obtained for the native enzyme. The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide. However, in the presence of fructose-6-phosphate (fru-6-P) and ATP−4, the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer. Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295. This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence. Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a Kd value that is 6-fold higher than the one determined for the native enzyme. These are the first residues to be implicated in the activity and/or structure of the Pfk-2.

scholarly journals Characterization of one new non-S-RNase of Armeniaca cathayana

2019 ◽  
Vol 55 (No. 1) ◽  
pp. 39-41
Author(s):  
Mengpei Liu ◽  
Pei Hou ◽  
Xiaoyuan Wang ◽  
Yu Dong ◽  
Wei Zong
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Self Incompatibility ◽  
Blast Analysis ◽  
New Information ◽  
S Allele ◽  
The One ◽  
A New Species ◽  
Prunus Webbii

Armeniaca cathayana, a new species described in 2010, belongs to gametophytic self-incompatibility (GSI) system which is under S-allele control. One new non-S-ribonuclease (non-S-RNase) was found in A. cathayana through comparing its nucleotide and amino acid sequences with sequences of the S-allele in Genbank. The BLAST analysis showed that the one new non-S-RNase S68-RNase (GenBank Accession No. MH155952) had the highest 96% nucleotide sequence homology with Prunus webbii non-S-RNase PW<sub>1</sub> (EU809938.1). Alignment of deduced amino acid sequences of A. cathayana S68-RNase shared 83% similarity with P. webbii PW<sub>1</sub>. The new non-S-RNase determined in this study will provide new information to GSI of Rosaceae.  

Genetic characterization of Spermophagus niger (Coleoptera: Chrysomelidae: Bruchinae: Amblycerini) pest associated to seeds of Sorrel (Hibiscus sabdariffa L.) in Burkina Faso

2016 ◽  
Vol 6 (1) ◽  
pp. 07-14
Author(s):  
Jean Christophe Koussoubé ◽  
Fatimata Mbaye ◽  
Cheikh Abdou Khadre Mbacké Dia ◽  
Mbacké Sembène ◽  
Antoine Sanon
Keyword(s):  
Amino Acid ◽  
Burkina Faso ◽  
Genetic Parameters ◽  
Genetic Characterization ◽  
Mitochondrial Gene ◽  
Hibiscus Sabdariffa ◽  
Polymerase Chain ◽  
The One ◽  
First Time

In Burkina Faso, the seeds of sorrel, Hibiscus sabdariffa L. are attacked by a pest identified morphologically as Spermophagus niger which is maintained all year on seeds and causing considerable damages. In the current study, for the first time, genetic characterization for S. niger was performed to determine its genetic identity and place it in its phyletic group. Mitochondrial gene, the Cytochrome oxidase I (COI) of the pest was partially sequenced after extraction and amplification by Polymerase Chain Reaction (PCR). Then the variability of genetic parameters namely the number of polymorphic and monomorphic sites, the frequencies of the different nucleotides and amino acid composition were determined. The nucleotide sequence of S. niger ob-tained was submitted in Genbank and the accession number is KU710716. Nucleotide sequences of S. niger obtained and those of different species of Spermophagus and Z. subfasciatus available in the GenBank database, we determined the percentage of similarity on the one hand and kinship through Phylogenetics reconstructions on the other hand. The results showed the absence of polymorphic sites for 406 sites obtained with 36.5% of thymine, 17.5% of cytosine, adenine 31% and 15% of guanine. Leucine was the majority amino acid (14.50%); the lysine was minority amino acid (0.76%) and cysteine was absent. The percentage of similarity obtained and phylogenetics reconstructions showed that S. niger is very close to the different species of Spermophagus particularly S. drak and different from Z. sub-fasciatus.

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