The First Crystal Structure of L-Threonine Dehydrogenase

Kazuhiko Ishikawaa, Noriko Higashia, 1, Tsutomu Nakamuraa, Takanori Matsuurab, and Atsushi Nakagawab

aNational Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
bInstitute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
1Present address: Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan

Journal of Molecular Biology, 336(3), 857-867 (2007)

L-Threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homotetramer structure with crystallographic and noncrystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the α/β Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits a coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.

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MATSUURA Takanori (Please change the mark “%” to “@”)

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