Structure and reactivity of intermediates in the carboligation of ThDP-dependent enzymes

ThDP依赖性酶的碳化过程中中间体的结构和反应性

• 批准号: 172044233
• 负责人: Professor Dr. Kai Tittmann
• 金额: --
• 依托单位: Göttinger Zentrum für Molekulare Biowissenschaften (GZMB)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/172044233?language=en
• 关键词: Structure; reactivity; intermediates; carboligation; ThDP

Besides their well-characterized functions in cellular metabolism, thiamine diphosphate (ThDP)-dependent enzymes have recently emerged as powerful tools for the enantio- and stereoselective carboligation of 2-ketoacids, aldehydes and sugars to yield a variety of pharmaceutically interesting compounds. A crucial issue for deciphenng the reaction and substrate specificity of carboligation relates to the analysis of molecular detenninants that define the binding sites and therewith specificity of donor and acceptor substrates, and the kinetic (de)-stabilisation of the central carbanion/enamine intermediate deriving from the donor. Exemplified for the prototypical ThDP-dependent enzymes pyruvate decarboxylase (PDC) and transketolase (TK), our studies are aimed to - on a molecular level - deduce mechanistic principles by which off-pathway (PDC) or on-pathway (TK) carboiigation are conferred. On the basis of available kinetic, spectroscopic and structural information, carboligation in wild-type enzymes and variants with putatively altered substrate binding pockets and a kinetically stabilized carbanion/enamine intermediate shall be rigorously examined by means of microscopic kinetic and thermodynamic analysis. When appropriate, key intermediates of carboligation will be structurally characterized by X-ray crystallography. In conclusion, a mechanistic framework shall be developed that allows to rationally design tailor-made ThDP-dependent enzymes with defined reaction and substrate specificity.

除了它们在细胞代谢中的良好表征的功能之外，硫胺素二磷酸（ThDP）依赖性酶最近已经成为用于2-酮酸、醛和糖的对映体和立体选择性碳化以产生各种药学上感兴趣的化合物的有力工具。一个关键的问题，decophenng的反应和底物特异性的carboligation涉及到分析的分子detenninants定义的结合位点和特异性的供体和受体底物，和动力学（去）-稳定的中心碳负离子/烯胺中间体衍生自供体。以原型ThDP依赖性酶丙酮酸脱羧酶（PDC）和转酮醇酶（TK）为例，我们的研究旨在在分子水平上推导出赋予途径外（PDC）或途径上（TK）碳水化合物连接的机制原理。在现有动力学、光谱和结构信息的基础上，应通过微观动力学和热力学分析，严格检查野生型酶和具有puronically改变的底物结合口袋和动力学稳定的碳负离子/烯胺中间体的变体中的碳结合。在适当的情况下，碳化的关键中间体将通过X射线晶体学进行结构表征。总之，应开发一种机制框架，其允许合理地设计具有限定的反应和底物特异性的定制ThDP依赖性酶。

项目成果

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

• DOI: 10.3390/catal6120190
• 发表时间: 2016-12-01
• 期刊: CATALYSTS
• 影响因子: 3.9
• 作者: Andrews，Forest H.;Wechsler，Cindy;McLeish，Michael J.
• 通讯作者: McLeish，Michael J.

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot

• DOI: 10.1002/cbic.201500529
• 发表时间: 2015-12
• 期刊: ChemBioChem
• 影响因子: 3.2
• 作者: Cindy Wechsler;Danilo Meyer;S. Loschonsky;L. Funk;P. Neumann;R. Ficner;Florian Brodhun;Michael Müller;K. Tittmann