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PEDS Advance Access originally published online on February 3, 2006
Protein Engineering Design and Selection 2006 19(4):163-167; 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

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

Y. Kai1, T. Kashiwagi1, K. Ishikawa1, M.K. Ziyatdinov2, E.I. Redkina2, M.Y. Kiriukhin2, M.M. Gusyatiner2, S. Kobayashi3, H. Takagi3 and E. Suzuki1,4

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

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

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

Received August 18, 2005; revised December 4, 2005; accepted January 3, 2006.

Edited by Haruki Nakamura


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