scholarly journals Purification and Properties of Arabidopsis thaliana COR (Cold-Regulated) Gene Polypeptides COR15am and COR6.6 Expressed in Escherichia coli

1996 ◽  
Vol 111 (1) ◽  
pp. 293-299 ◽  
Author(s):  
S. J. Glimour ◽  
C. Lin ◽  
M. F. Thomashow
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Purification And Properties

scholarly journals Purification and properties of cystathionine β-lyase from Arabidopsis thaliana overexpressed in Escherichia coli

1996 ◽  
Vol 320 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Stéphane RAVANEL ◽  
Dominique JOB ◽  
Roland DOUCE
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Biochemical Properties ◽  
Coli Strain ◽  
High Yield ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Schiff Base Formation ◽  
Insight Into ◽  
Base Formation

Cystathionine β-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5´-phosphate-dependent β-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine β-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine β-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5´-phosphate. Interaction between the apoenzyme and pyridoxal 5´-phosphate is extremely tight, being characterized by a Kd value of 0.5 µM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine β-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine β-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5´-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.

scholarly journals Purification and Properties of Phosphatidylserine Decarboxylase from Escherichia coli

1974 ◽  
Vol 249 (10) ◽  
pp. 3079-3084 ◽  
Author(s):  
William Dowhan ◽  
William T. Wickner ◽  
Eugene P. Kennedy
Keyword(s):  
Escherichia Coli ◽  
Purification And Properties
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