Publications

• Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli.

  Holbrook E.L., Greene R.C., Krueger J.H.

  To characterize the methionine biosynthetic enzyme cystathionine gamma-synthase from Escherichia coli, we have constructed high copy number plasmids containing the metB structural gene but lacking the closely linked metJ regulatory gene. When cloned into an appropriate strain, these plasmids can d ... >> More

  To characterize the methionine biosynthetic enzyme cystathionine gamma-synthase from Escherichia coli, we have constructed high copy number plasmids containing the metB structural gene but lacking the closely linked metJ regulatory gene. When cloned into an appropriate strain, these plasmids can direct the overproduction of cystathionine gamma-synthase such that about 10% of the soluble protein is this enzyme. An efficient purification scheme has been developed that has allowed us to obtain gram quantities of enzyme. The active form is a tetramer with subunits of about 40,000 daltons and one pyridoxal phosphate cofactor per monomer. The kinetic constants for several enzyme-catalyzed reactions were determined at 25 degrees C. The Km value for the elimination reaction with O-succinyl-L-homoserine was calculated to be 0.33 mM with maximal velocity of 460 min-1. The Km for the elimination (deamination) reaction with vinylglycine was 5.6 mM with maximal velocity of 900 min-1. The Km values for the replacement reaction were calculated to be 1.0 mM for O-succinyl-L-homoserine and 0.05 mM for L-cysteine with maximal velocity of 700 min-1. The enzyme shows an absorption band at 422 nm (epsilon = 8463 M-1 cm-1) attributable to the Schiff base form of the pyridoxal phosphate cofactor. Steady-state spectra of reaction complexes show appearance of new longer wavelength absorbing materials during reaction with O-succinyl-L-homoserine, vinylglycine, or vinylglycine and L-cysteine. Reaction with O-succinyl-L-homoserine and L-cysteine produces only a red shift and slight reduction of the band at 422 nm. << Less

  Biochemistry 29:435-442(1990) [PubMed] [EuropePMC]

• Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli.

  Ravanel S., Gakiere B., Job D., Douce R.

  Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overe ... >> More

  Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 microM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma-elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme. << Less

  Biochem. J. 331:639-648(1998) [PubMed] [EuropePMC]

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