Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum.
Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-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 competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.
Clausen
T
T
Max-Planck-Institut für Biochemie, Planegg-Martinsried, Germany.
Wahl
M C
MC
Messerschmidt
A
A
Huber
R
R
Fuhrmann
J C
JC
Laber
B
B
Streber
W
W
Steegborn
C
C
D000595:Amino Acid Sequence; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases