单酶催化多步连续反应中底物结合模式研究

In biological metabolism，some generalist enzymes can catalyze multi-step sequential reactions, for instance，the representative desaturase CrtI converts phytoene to neurosporene, lycopene, and other desaturation products in alternative multi-step sequential dehydrogenations. If the interactions features between enzyme and the each step substrate are clear in the multi-step sequential reactions catalyzed by a single enzyme, and such multi-step sequential reactions mechanism could be explained, which will promote the contributions of the generalist enzymes to building artificial cells. The applicant proposes the hypothesis of two substrate-binding modes, shift mode and transformation mode. Subsequently the applicant plans to study the substrate-binding mode of the generalist enzyme in the multi-step sequential reactions, by means of combining the simulation of complex structure of enzyme and the each step substrate, with the enzyme reaction kinetics assays. In this study, we will use CrtI from Blakeslea trispora as the study object, model complex structures of CrtI and the each step substrate; analyze enzyme reaction kinetics of wild-type CrtI and mutants in vitro; further optimize and comparative analyze the complex structures of enzyme and the each step substrate, to clarify the substrate-binding mode of CrtI in multi-step sequential dehydrogenations. This study will further perceive the substrate-binding of enzyme in multi-step sequential reactions catalyzed by a single enzyme, and supply valuable references for studying the product inhibition of enzyme catalysis and the multi-step sequential reactions mechanism.

在生物代谢过程中存在一类可催化多步连续反应的通用酶，例如脱氢酶CrtI可以催化八氢番茄红素发生多步连续脱氢反应依次生成链孢红素和番茄红素等多种产物。若能够明确单酶催化多步连续反应中各步反应底物与酶的结合机制，就能理解单酶催化多步连续反应机制，促进该类酶在人工细胞构建中的贡献。申请人设想从位移模式和形变模式入手，通过酶-底物复合体结构模拟分析和体外酶反应动力学测定相结合的技术手段，阐明各步反应底物与酶的结合机制，进而研究多步连续反应中酶与底物的结合模式。本项目以脱氢酶CrtI为例，构建CrtI与各步反应底物复合体的结构模型，表征CrtI及其关键突变体的体外酶反应动力学特征。在此基础上比较分析酶与底物复合体的结构模型和结合机制，阐明多步连续反应中酶与底物的结合模式。本项目将创新对单酶催化多步连续反应中酶结合各步底物的认知，为研究酶的产物抑制作用和多步连续反应机制提供了重要的参考依据。

脱氢酶CrtI可以催化多步连续脱氢反应，不同物种的脱氢反应步骤不同。然而，控制脱氢步骤的结构特征尚不清楚。若能够明确单酶催化多步连续反应中各步反应底物与酶的结合机制，就能理解单酶催化多步连续反应机制，促进该类酶在人工细胞构建中的贡献。申请人设想从位移模式和形变模式入手，通过酶-底物复合体结构模拟分析和酶功能表征相结合的技术手段，阐明各步反应底物与酶的结合机制，进而研究多步连续反应中酶与底物的结合模式。本项目利用定向进化技术模拟自然进化过程，对四步脱氢酶BtCrtI的脱氢功能的改变，确定了两个阳性突变体H136R和H453R，使脱氢反应步骤分别减少为3步和2步。通过序列比对获得了关键突变位点系统进化分析的信息。结合CrtI与底物复合体的结构模型以及CrtI关键突变体的催化反应特异性，阐明这类酶进化过程的构效关系。此外，我们考察了BtCrtI的H136和H453的饱和突变结果，并采用主成分分析(PCA)和k-均值算法对结果进行聚类统计。本研究确定了调控BtCrtI脱氢步骤的关键位点，并提出了相关的结构进化假说，为酶进化理论提供了新的内容。