The elucidation of molecule evolution with respect to chiral recognition of enzymes.

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14560286
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied molecular and cellular biology
• Research Institution: University of Yamanashi
• Principal Investigator: UI Sadaharu Univ. of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Professor, 大学院・医学工学総合研究部, 教授 (80115310)
• Co-Investigator(Kenkyū-buntansha): KUSUNOKI Masami Osaka Univ., Institute for protein Research, Associate Professor, 蛋白質研究所, 助教授 (90135749)
• Project Period (FY): 2002 – 2003
• Keywords: 2,3-butanediol / acetoin / 2,3-butanediol dehydrogenase / stereospecificity / chiral / SDR family / X-ray crystallography / protein engineering

Research Abstract

The research contents : It was clarified that meso-2,3-butanediol dehydrogenase (BDH) and L-BDH are short chain dehydrogenase / reductase (SDR) enzymes and have a similar structure. Then, using the structural knowledge of these enzymes, the elucidation of molecule evolution, with respect to enzyme functions such as stereospecificity and stability, was attempted. Various BDH-mutants were prepared by exchanging the domains and the amino acids in the active sites between meso-BDH and L-BDH. Next, crystal structure analyses of some of the mutants were performed. In additions, the influence on the stereo-structure of various mutations was analyzed by molecule calculation techniques. Furthermore, the influences of the enzyme structure on kinetic parameters were analyzed. Based on the results, the molecule evolution between L-BDH and meso-BDH was considered from the standpoint of an enzyme function.
Result : Interesting results were obtained mainly on following: 1)Amino acid residues related t o Km value, 2)Amino acid residues related to Stereospecificity, 3)Analysis of the recognition mechanism for long-chain substrates, 4)Analysis of the dehydrogenase reaction mechanism in 3R-configulation of substrate, 5)Analysis of the substrate recognition mechanism from the competitive-inhibition constant of 2-mercaptoethanol, 6)Analysis of the influence of the mutation-point on Vmax, and 7)Analysis of the structure contributing to stability. Using the elucidated information, the characteristics of the enzyme have been improved regarding structure. That is, unstable wild-type L-BDH was successfully changed to a new L-BDH with high stability. Moreover, these results offered a fundamental indicator for the appropriate alteration to other SDR enzymes having a structure similar to that of BDH.
The production and manufacture methods of L-BD were developed using the L-BDH with newly created high stability. The efficient production of L-BD was not established until now. That is, production on the thousands of mg/l level was attained at about an 80% conversion rate from diacetyl as substrate by expressing the meso-BDH and the mutant-L-BDH simultaneously in transgenic E. coli.
At this time, the above results are under contribution as some reports.