Thermal stability, function and dynamics of an euzyme

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 04044068
• Research Category: Grant-in-Aid for international Scientific Research
• Allocation Type: Single-year Grants
• Section: Joint Research
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: OSHIMA Tairo Tokyo Institute of Technology, Dept.of Life Science, 生命理工学部, 教授 (60167301)
• Co-Investigator(Kenkyū-buntansha): ARISAKA Fumio Tokyo Institute of Technology, Dept.of Life Science, 生命理工学部, 助教授 (80133768) ; TANAKA Nobuo Tokyo Institute of Technology, Dept.of Life Science, 生命理工学部, 教授 (50032024) ; KATSUBE Yukiteru Osaka University, Broteiu Research Inst., 蛋白質研究所, 名誉教授 ; ZAVODSZKY Peter Hungary Academy of Sci., Inst.for Euzymology, 教授 ; PETSKO Gregory Brandeis University, Dept.of Chemistry, 化学科, 教授
• Project Period (FY): 1992 – 1994
• Keywords: Thermophile / Hyperthermophile / Stabilization of Enzyme / Evolutional Molecular Engineering / Thermus thermoplulus / 超好熱菌

Research Abstract

Using 3-isopropylmalate dehydrogenase as a model enzyme protein, our international and interdisciplinary team studied the relationship between three dimensional structure, stability and function by different experimental approaches. Japanese members designed, purified, and crystallized the thermophile enzyme and its mutants, and distributed these enzyme proteins among the team members.
1 An experimental system for laboratory evolution using an extreme thermophile, Thermus thermophilus, as the host has been invented. Using this system, we attempted and succeeded to stabilize a chimeric 3-isopropylmalate dehydrogenase. The genetic analysis of the stabilized mutants showed that Ala172Val replacement is key for the increased stability of one of the stabilized chimeras. The detailed x-ray analysis suggested that the gap around the side chain of Ala-172 is filled with this replacement and the increased stability was conferred by this gap filling.
2 Crystallographic analyzes suggested that the stability of the chimera can be improved by stabilizing a loop around residue-110. The site-directed mutagenesis studies proved that the combination of two factors, release of constrain by Gly residue and additional hydrogen bonding in the loop, is essential for the improving the stability of the chimera.
3 3-Isopropylmalate dehydrogenase from a hyperthermophile was purified and characterized. The enzyme is more stable than the corresponding one from T.thermophilus. The enzyme protein was crystallized and the crystals were distributed among the team members.