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

Abstract

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.

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

“…2) (39), the deduced amino acid sequence of CYS3 has highly significant homology with several enzymes involved in metabolism of sulfur-containing amino acids; E. coli CT 1y-synthase (8), ClT l-lyase (9), rat CIT ly-lyase (11), and S. cerevisiae OAS-OAH sulfhydrylase (6,24). All these proteins are known to have oligomeric structures composed of identical subunits of molecular weights ranging from 40,000 to 50,000 and to require PLP as a cofactor (11,17,44,47). No Figure 3 also shows the result of SDS-PAGE of the purified preparation of CYS3 gene product (lane P), indicating that the preparation obtained has a purity of 90% or higher.…”

Molecular characterization of Mycobacterium tuberculosis cystathionine gamma synthase—Apo- and holoforms

“…In trans-sulphuration pathway, l-cysteine condenses with activated l-homoserine to form l-cystathionine (CTH) in the presence of PLP-dependent (pyridoxal 5 phosphate dependent) cystathionine ␥-synthase (CGS). This intermediate lcystathionine is then split asymmetrically into l-homocysteine and pyruvate by cystathionine ␤-lyase followed by methylation of l-homocysteine yielding l-methionine [5,6]. In reverse trans-sulphuration pathway, CGS generates l-cystathionine, from l-homocysteine and l-serine, which is acted upon by cystathionine gamma lyase (CGL) to form l-cysteine, ␣-ketobutyrate and ammonia [7].…”

“…→ l-cystathionine + succinate (1) O-succinyl-l-homoserine + H 2 O →˛-ketobutyrate + ammonia + succinate (2) Most studies on this enzyme have been done on Salmonella typhimurium [5,10], Escherichia coli [6,11], Arabidopsis thaliana [12], wheat [13] and spinach [14]. Though the enzymes from bacteria and plants use different substrates, i.e.…”

Cloning and analysis of the gene for cystathionine ?-synthase from Arabidopsis thaliana

“…This enzyme will catalyze a ~y-elimination reaction in the absence of Cys to yield succinate, c~-ketobutyrate and ammonia [ 14]. The plant enzyme very likely uses OPH as the physiological substrate, but it can also use a variety of homoserine esters including OSH [4,9].…”