Purification and properties of cystathionine beta-lyase from Arabidopsis thaliana overexpressed in Escherichia coli.
Cystathionine beta-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5'-phosphate-dependent beta-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine beta-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 beta-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 microM. 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 beta-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 beta-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.
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Laboratoire Mixte CNRS/Rhône-Poulenc (UMR41), Rhône-Poulenc Agrochimie, Lyon, France.
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D018515:Plant Leaves; D016133:Polymerase Chain Reaction; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D018076:DNA, Complementary; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D008190:Lyases; C019965:cystathionine beta-lyase