Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy |
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Authors: | Hiroki Yamaguchi Akiko Kamegawa Kunio Nakata Tatsuki Kashiwagi Toshimi Mizukoshi Yoshinori Fujiyoshi Kazutoshi Tani |
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Institution: | 1. Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki 210-8681, Japan;2. Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan;3. CeSPIA Inc., 2-1-1, Otemachi, Chiyoda, Tokyo 100-0004, Japan;4. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo, Japan |
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Abstract: | Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2?Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80?°C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability. |
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Keywords: | FSC fourier shell correlation PDB Protein Data Bank RMSD root-mean-square deviation XRD X-ray diffraction ELFV Glu/Leu/Phe/Val BCAA cryo-EM cryo-electron microscopy bGDH bovine glutamate dehydrogenase LDH leucine dehydrogenase Conformational change Single particle analysis De novo modeling Cryo-EM |
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