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Protein Engineering vol. 8 no. 8 pp. 763-767, 1995
© 1995 Oxford University Press

RESEARCH-ARTICLE

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

Masahiro Sakurai, Hideaki Moriyama1, Ko Onodera, Shojiro Kadono, Koichi Numata, Yoko Hayashi, Jitsutaro Kawaguchi, AkJhiro Yamagishi, Tairo Oshima and Nobuo Tanaka

Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan

1To whom correspondence should be addressed

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.

Keywords: chimera/crystal structure/3-isopropylmalatedehydrogenase/site-directed mutagenesis

Received January 27, 1995; revised May 19, 1995; accepted June 6, 1995.


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