Molecular Mechanism of Stereospecific Enzyme Reactions

Research Project

Project/Area Number	07456049
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Research Field	応用微生物学・応用生物化学
Research Institution	Nara Institute of Science and Technology
Principal Investigator	HASHIMOTO Takashi (1997) NAIST,Biological Sciences, Associate Professor, バイオサイエンス研究科, 助教授 (80180826) 山田康之 (1995-1996) 奈良先端科学技術大学院大学, バイオサイエンス研究科, 教授 (50026415)
Co-Investigator(Kenkyū-buntansha)	NAKAJIMA Keiji NAIST,Biological Sciences, Research Associate, バイオサイエンス研究科, 助手 (80273853) 西岡孝明京都大学, 農薬研究施設, 教授 (80026559) 橋本隆奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教授 (80180826)
Project Period (FY)	1995 – 1997
Project Status	Completed (Fiscal Year 1997)
Budget Amount *help	¥8,400,000 (Direct Cost: ¥8,400,000) Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000) Fiscal Year 1996: ¥2,400,000 (Direct Cost: ¥2,400,000) Fiscal Year 1995: ¥4,500,000 (Direct Cost: ¥4,500,000)
Keywords	stereospecificity / crystal structure / reductase / tropinone / alkaloid / stereospecificity / crystallography / reductase / alkaloid
Research Abstract	A pair of tropinone reductase (TRs) share 64% identical amino acid residues, and belong to the short-chain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups having different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-* resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains which are conserved within the protein family, but also extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the potitions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate-binding site, thereby determining the stereospecificity of the reaction product. The results obtained raised the possibility that in certain cases novel stereospecificities can be acquired in emzymes by changing a few amino acid residues within substrate-binding sites.