The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T.
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 chimeric 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 A resolution. Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain. The R-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. Denaturation by heating may be interpreted from these structural results.
Sakurai
M
M
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Moriyama
H
H
Onodera
K
K
Kadono
S
S
Numata
K
K
Hayashi
Y
Y
Kawaguchi
J
J
Yamagishi
A
A
Oshima
T
T
Tanaka
N
N
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D007532:Isoleucine; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D012694:Serine; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase