Cloning, purification and characterisation of cystathionine gamma-synthasefrom Nicotiana tabacum

Cystathionine gamma-synthase, the enzyme catalysing the first reaction spec ific for methionine biosynthesis, has been cloned from Nicotiana tabacum, o verexpressed in Escherichia coli and purified to homogeneity, The recombina nt cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate depend ent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent K-m-values of 7.1 +/- 3.1 mM and of 0.23 +/- 0.07 mM, respec tively, The enzyme was irreversibly inhibited by DL-propargylglycine (K-i = 18 mu M, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogue s 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridin e-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competit ive inhibitors with K-i values of 0.20, 0.30, 0.45, and 0.027 mM, respectiv ely, In combination these results suggest a ping-pong mechanism for the cys tathionine gamma-synthase reaction, with homoserine phosphate binding to th e enzyme first, Large single crystals of cystathionine y-synthase diffracti ng to beyond 2.7 Angstrom resolution were obtained by the sitting drop vapo ur diffusion method, The crystals belong to the orthorhombic space group P2 (1)2(1)2(1) with unit cell constants a = 120.0 Angstrom, b = 129.5 Angstrom , c = 309.8 Angstrom, corresponding to two tetramers per asymmetric unit.