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

阐明酶的手性识别方面的分子进化。

• 批准号: 14560286
• 负责人: UI Sadaharu
• 金额: $ 2.24万
• 依托单位: University of Yamanashi
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560286/
• 关键词: 2,3-butanediol; acetoin; 2,3-butanediol dehydrogenase; stereospecificity; chiral; SDR family; X-ray crystallography; protein engineering; short chain dehydrogenase; reductase family; X-ray crystallography; shrt chain dehydrogenase

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 … More 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. Less

研究内容：明确了中位2,3-丁二醇脱氢酶（meso-2,3- butyanediol dehydrogenase， BDH）和L-BDH都是短链脱氢酶/还原酶（SDR），具有相似的结构。然后，利用这些酶的结构知识，试图阐明分子进化，关于酶的功能，如立体特异性和稳定性。通过交换中位bdh和L-BDH活性位点的结构域和氨基酸，制备了多种bdh突变体。接下来，对一些突变体进行晶体结构分析。此外，利用分子计算技术分析了各种突变对立体结构的影响。进一步分析了酶的结构对动力学参数的影响。在此基础上，从酶功能的角度考虑了L-BDH与中位bdh之间的分子演化过程。结果：有趣的结果主要体现在以下几个方面：1)与…More o Km值相关的氨基酸残基，2)与立体特异性相关的氨基酸残基，3)对长链底物的识别机制分析，4)对底物3r构型脱氢酶反应机制分析，5)从2-巯基乙醇的竞争抑制常数分析底物识别机制，6)突变点对Vmax的影响分析，7)对稳定性的结构分析。利用所解析的信息，改进了酶的结构特征。即将不稳定的野生型L-BDH成功转化为具有高稳定性的新型L-BDH。此外，这些结果为适当改变其他与BDH结构相似的SDR酶提供了基础指标。利用新研制的高稳定性L-BDH，开发了L-BDH的生产和制造方法。L-BD的高效生产至今尚未建立。也就是说，通过在转基因大肠杆菌中同时表达中位bdh和突变型l - bdh，以双乙酰基为底物的转化率达到了数千mg/l的水平。此时，上述结果正在作为部分报告进行贡献。少