THE CRYSTAL-STRUCTURE OF THERMOSTABLE MUTANTS OF CHIMERIC 3-ISOPROPYLMALATE DEHYDROGENASE, 2T2M6T

A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produc ed by replacing the amino acid sequences of the Thermus thermophilus e nzyme with those of the Bacillus subtilis enzyme from residues 75 to 1 13, Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S 82R), The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 Angstrom resolution, Although S82R was refined rout inely, I93L required the preliminary rigid-body refinement of each dom ain, The R-factors were reduced to 0.18 for both mutants, Removal of t he unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T, In the case of S82R, the replaced arginine r esidue contributed to the extra hydrogen bond with water molecules, Th e large replaced residue decreased the entropy of the solvent, which m ay have caused the improvement in enzyme thermostability. Denaturation by heating may be interpreted from these structural results.