Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 19, Issue 4, Pages 163-167Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzj015
Keywords
desensitization; Escherichia coli; feedback inhibition; L-cysteine; L-serine O-acetyltransferase
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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 C alpha 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; IC50 = 1100 mu mol/l, the inhibition constant; K-i = 950.0 mu mol/l) and extremely high emzymatic activities.
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