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PEDS Advance Access published online on February 3, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzj015
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received August 18, 2005
Revised December 4, 2005
Accepted January 3, 2006

Article

Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine

Y. Kai 1, T. Kashiwagi 1, K. Ishikawa 1, M. K. Ziyatdinov 2, E. I. Redkina 2, M. Y. Kiriukhin 2, M. M. Gusyatiner 2, S. Kobayashi 3, H. Takagi 3, and E. Suzuki 1 *

1 Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan
2 Ajinomoto-Genetika Research Institute, Moscow 117545, Russia
3 Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan

* To whom correspondence should be addressed.
E. Suzuki, E-mail: eiichiro_suzuki{at}ajinomoto.com


  Abstract

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 µmol/l, the inhibition constant; Ki = 950.0 µmol/l) and extremely high emzymatic activities.

Keywords: desensitization; Escherichia coli; feedback inhibition; L-cysteine; L-serine O-acetyltransferase.
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