Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine.
L-Serine O-acetyltransferase (SAT) from Escherichia coli catalyzes the first step of L-cysteine synthesis in E.coli and is strictly inhibited by the second step product, L-cysteine. To establish a fermentation process to produce L-cysteine, we embarked on a mutational study of E.coli SAT to desensitize the feedback inhibition by L-cysteine. The crystal structure and the reaction mechanism of SAT from E.coli have shown that the substrate L-serine and the inhibitor L-cysteine bind to the identical region in the SAT protein. To decrease the affinity for only L-cysteine, we first built the structure model of L-serine-binding SAT on the basis of the crystal structure with bound L-cysteine and compared these two structures. The comparison showed that the Calpha of Asp92 underwent a substantial positional change upon the replacement of L-cysteine by L-serine. We then introduced various amino acid substitutions at positions 89-96 around Asp92 by randomized, fragment-directed mutagenesis to change the position of the Asp92. As a result, we successfully obtained mutant SATs which have both extreme insensitivity to an inhibition by L-cysteine (the concentration that inhibits 50% activity; IC(50) = 1,100 micromol/l, the inhibition constant; K(i) = 950.0 micromol/l) and extremely high emzymatic activities.
Kai
Y
Y
Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki 210-8681, Japan.
Kashiwagi
T
T
Ishikawa
K
K
Ziyatdinov
M K
MK
Redkina
E I
EI
Kiriukhin
M Y
MY
Gusyatiner
M M
MM
Kobayashi
S
S
Takagi
H
H
Suzuki
E
E
D019943:Amino Acid Substitution; D003545:Cysteine / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D025461:Feedback, Physiological / Q000187:drug effects; D020128:Inhibitory Concentration 50; D051050:Serine O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*
D051050:Serine O-Acetyltransferase; D003545:Cysteine