Cloning, purification, and characterization of beta-cystathionase from Escherichia coli.
The Clarke-Carbon clone bank of hybrid plasmid Escherichia coli DNA has been screened for plasmids able to complement an E. coli strain deficient for the production of beta-cystathionase. Clone 4-14 had the ability to complement a deletion mutation at this locus and expressed higher levels of beta-cystathionase than the wild-type strain. The transfer of the plasmid carried by this clone to a strain that constitutively expresses all the enzymes of the methionine biosynthetic pathway results in 100-fold overproduction of beta-cystathionase as compared to wild-type levels. With use of this strain, an efficient three-step purification scheme is described that gives 90% pure enzyme in 54% yield with a specific activity of 215 IU/mg. This enzyme is characterized as to molecular weight (280 000), number of subunits (six), pyridoxal phosphate binding (5.7 mol of pyridoxal phosphate bound/mol of protein, Km of 0.005 mM), amino acid composition, substrate specificity, and kinetic properties.
D000596:Amino Acids / Q000032:analysis; D003001:Cloning, Molecular; D003540:Cystathionine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D006710:Homocysteine / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000235:genetics*; D008970:Molecular Weight; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D000596:Amino Acids; D006710:Homocysteine; D003540:Cystathionine; D011732:Pyridoxal Phosphate; D008190:Lyases; C019965:cystathionine beta-lyase