The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T

Masahiro Sakurai, Hideaki Moriyama, Ko Onodera, Shojiro Kadono, Koichi Numata, Yoko Hayashi, Jitsutaro Kawaguchi, Akjhiro Yamagishi, Tairo Oshima, Nobuo Tanaka

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12 Scopus citations


A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113. Decreased thermostability of the chiaieric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R). The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 Å resolution. Although S82R was refined routinely, (I93L) required the preliminary rigid-body refinement of each domain. The X-factors were reduced to 0.18 for both mutants. Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T. In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules. The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability. Denatu ration by heating may be interpreted from these structural results.

Original languageEnglish (US)
Pages (from-to)763-767
Number of pages5
JournalProtein Engineering, Design and Selection
Issue number8
StatePublished - Aug 1995
Externally publishedYes


  • 3-isopropylmalatedehydrogenase
  • Chimera
  • Crystal structure
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology


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