Engineering of Escherichia colil-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by l-cysteine
Protein Engineering, Design & Selection
Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine
Y.Kai 2 3
T.Kashiwagi 2 3
K.Ishikawa 2 3
M.K.Ziyatdinov 1 2
E.I.Redkina 1 2
M.Y.Kiriukhin 1 2
M.M.Gusyatiner 1 2
S.Kobayashi 0 2
H.Takagi 0 2
E.Suzuki 2 3
0 Department of Bioscience, Fukui Prefectural University , Fukui 910-1195 , Japan
1 Ajinomoto-Genetika Research Institute , Moscow 117545 , Russia
2 The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions , please
3 Institute of Life Sciences, Ajinomoto Co., Inc. , 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681 , Japan
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 Ca 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 lmol/l, the inhibition constant; Ki = 950.0 lmol/l) and extremely high emzymatic activities.
Introduction
L-Cysteine plays crucial roles in the structure, stability and
catalytic function of many proteins, and is also an important
amino acid used in the pharmaceutical, food and cosmetics
industries. In enteric bacteria such as Escherichia coli and
Salmonella typhimurium
(Kredich and Tomkins, 1966;
Kredich et al., 1969; Soda, 1987)
and higher plants
(Smith
and Thompson, 1969; Nakamura et al., 1987)
, L-cysteine is
synthesized via O-acetyl-L-serine through the pathway
constituted with L-serine O-acetyltransferase (SAT) (EC 2.3.1.30)
and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8). The SAT
enzyme catalyses the formation of O-acetylation from
acetylCoA and L-serine, which is the first reaction in the two-step
process of sulfur assimilation.
L-serine + acetyl-CoA!O-acetyl-L-serine + CoA
ð1Þ
ð2Þ
High-level production of L-cysteine from glucose has not been
achieved successfully in microorganisms because of the
feedback inhibition of SAT by L-cysteine
(Kredich and Tomkins,
1966; Kredich et al., 1969)
. Takagi et al. (1999a,b) have
recently reported that L-cysteine is overproduced to some
extent in the L-cysteine non-utilizing E.coli strains that express
the feedback inhibition-insensitive SATs (Nakamori et al.,
1998). Site-directed and PCR random mutagenesis in the
cysE gene encoding E.coli SAT was employed to construct
the mutant enzymes that cause L-cysteine overproduction
as a result of desensitization to feedback inhibition
(Nakamori et al., 1998; Takagi et al., 1999b)
. Expression
of two complementary DNAs encoding feedback
inhibitioninsensitive SAT isoforms of Arabidopsis thaliana
(Noji et al., 1998)
also resulted in L-cysteine overproduction
(Takagi et al., 1999a)
. However, these SATs showed
significant decreases in enzymatic activity relative to the E.coli
wild-type enzyme (feedback inhibition-sensitive). Further
improvements in L-cysteine production are therefore expected
to use an engineered SAT, which shows a higher level of
feedback desensitization and a higher catalytic activity.
Recently, the three-dimensional (3D) structure of SAT from
E.coli with its inhibitor L-cysteine was determined by X-ray
crystallography
(Pye et al., 2004)
. This crystal structure was
solved by single-wavelength anomalous dispersion phasing
from seleno-L-methionine substituted SAT at 2.2 A˚ resolution.
SAT was shown to be a trimeric structure with 3-fold
symmetry, independent of the crystallographic 3-fold symmetry,
forming a three-sided pyramid shape. This trimer is likely to
interact with another SAT trimer at N-terminal ends. An
L-cysteine molecule was observed to bind at the serine
substrate site but not at the acetyl-CoA site between monomers
in the trimeric interaction.
In contrast,
Hindson and Shaw (2003)
have performed
steady-state kinetic and dead-end inhibition studies for
alternative substrates, which showed that the reaction
mechanism of SAT is a random-order ternary complex mechanism.
Hindson also showed that L-cystein (...truncated)