Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, re spectively) to the C-terminal region of the truncated enzyme. Three structu res of these mutant enzymes were determined by an X-ray diffraction method, All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stabilit y of these mutant enzymes is discussed based on the 3D structure with speci al attention to the width of the active-site groove and the minor groove, d istortion of beta-sheet pillar structure and size of cavity in the domain-d omain interface around the C-terminus. Our previous studies revealed that t he thermal stability of isopropylmalate dehydrogenase increases when the ac tive-site cleft is closed (the closed form). In the present study it is sho wn that the active-site cleft can be regulated by open-close movement of th e minor groove located at the opposite side to the active-site groove on th e same subunit, through a paperclip-like motion.