60NISR Research GRANT
2007 Young Investigator Research Grant

Mechanism of concerted inhibition of aspartate kinase fromCorynebacterium glutamicum
Takeo TOMITA
Biotechnology Research Center, The University of Tokyo

Research aims
A large amount of lysine is used for the food additives
for the livestock today, and lysine is produced by fermentation of high lysine-producing Corynebacterium glutamicum
mutants. The mutants were obtained by selecting for resistance to (S)-(aminoethyl)-L-cysteine (AEC), which is ananalogue of lysine, and the mutations are found in the regulatory domain of aspartate kinase (CgAK). The wild-type
CgAK is known to be susceptive to allosteric inhibition bysimultaneous addition of lysine and threonine in a concerted manner. Although CgAK is an attractive target in industrial and scientific fields, the regulatory mechanism of
CgAK remains to be elucidated. Thus, aim of this researchis to elucidate the mechanism of concerted inhibition ofCgAK and to obtain basic knowledge for the higher production of lysine or other amino acids.

added to the sample.
The crystal structure of CgAK was determined at 2.50 Åresolution. CgAK has a a 2b 2-type heterotetramer structure(Fig. 2). Two Thr and one Lys molecules are bound in twoeffector-binding units composed of b -subunit and regulatory domain of a -subunit, which has the sequence identicalto that of b -subunit (Four Thr molecules and two Lys molecules in whole structure). Thr is bound to the effector-binding unit in a manner very similar to that seen in CgAKbstructure. Among the two possible Lys-binding sites per ab
dimer, a Lys molecule is present in the binding site that isaway from the active site. The analyses of Lys/Thr inhibition profiles of the site-directed CgAK mutants carrying
single amino acid replacement at either of the two possible
binding sites showed that the only the Lys-binding site

Methods
Full length CgAK with a 2b 2 structure and its b -subunit(CgAKb ) were expressed in E. coli BL21 RIL-Codon Plus
(DE3) and those were purified with Ni-NTA affinity chromatography followed by gel filtration chromatography.
Through extensive screening for crystallization conditions,crystals with good diffraction were successfully obtainedfor both proteins. The structure determination of CgAKb
was performed by molecular replacement method (MR)using the structure of AKb from Thermus thermophilus,
which we determined previously by MAD method, as asearch model. The structure of CgAK was determined by
MR using the structure of AK from Methanococcus jannaschii and CgAKb as search models.

Fig. 1. Structure of CgAKb .
CgAKb forms a dimer of the two repeat peptide of ACT domain which isconsisted of two repeat of babbab -fold. The distinct unit (ACT1 andACT2) forms a interface of ligand binding which is novel manner Thr wasburied in the interface between the monomers.

Results
The crystal structure of CgAKb was determined at
1.58 Å resolution. CgAKb consists of a homodimer ofpolypeptides each containing two ACT (aspartate kinase,chorismate mutase, TyrA) domain (Fig. 1). CgAKb has aunique dimeric form in which two ACT domains each fromdifferent chain form a single effector-binding unit. As seenin the regulatory domain of AKs, a single subunit has aunique b -babba topology. Two Thr molecules are found in
the dimer interface in a manner where each of two effectorbinding unit binds a single Thr molecule. However, Lys was
not observed in the crystal structure in spite that it wasFig. 2. Structure of CgAK.
CgAK forms a interface between two Structure of a -subunits at the centerof the overall structure and b -subunits forms interfaces with distinct a subunits.

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residues, which is transmitted through the new interactions
between regulatory domain and catalytic domain.
The amino acid replacements which provide AEC resistance are mapped in CgAKb structure. Unexpectedly, thosemutations (A30V, S52F, and T59I) are clustered around theThr-binding site in spite of the fact that AEC is an analogof Lys (Fig. 3). Detailed analyses of the Lys/Thr inhibition
profiles indicated that A30V and T59I are responsible for
Thr-resitance and that S52F is involved in Lys-resistance,suggesting that former two residues are involved in the direct interaction with Thr and the latter is involved in thetransduction of the signal of Thr-binding to active sitethrough Lys-binding.
Fig. 3.

Mapping of AEC resistant mutation.

Conclusion
found in CgAK structure was functional in allosteric regulation. Comparison of the structures between E. coli AK inan active form and CgAK revealed that not only residues
involved in aspartate binding and but also those in ATPbinding are hampered in CgAK structure, suggesting thatThr/Lys binding induces allosteric conformational changeof the active site. Based on a structural comparison between
CgAK and CgAKb , it was suggested that the allosteric regulation proceeds with three steps; (i) Thr-induced association between of b -subunit and C-terminal regulatory domain of a -subunit, (ii) change in domain interaction by
Lys-binding, (iii) conformational change of the active site

The structures of AK from C. glutamicum complexedwith Lys and Thr and b -subunit of CgAK complexed withThr were determined. The structures shed a light to elucidate the mechanism of feedback inhibition of AK. This
study would provide the basic knowledge for the design of
new feedback-resistant enzymes.
References
1)

Yoshida, A., Tomita, T., Kurihara, T., Fushinobu, S., Kuzuyama, T.,and Nishiyama, M. (2007) Structural insight into concerted inhibition of a 2 b 2-type aspartate kinase from Corynebacterium glutamicum. J. Mol. Biol. 368, 521–536.