Structure and function of fumarate-adding glycyl radical enzymes: biochemistry, modelling and application

本提案旨在研究一类新型酶的生物化学和反应机制，这些酶催化富马酸根基加成到一些高顽固性化合物，如碳氢化合物。这些富马酸添加酶（FAE）是甘酰基自由基酶的一个亚类，对氧高度敏感，需要通过特殊的激活酶激活到自由基状态。拟议的项目是德国和波兰合作伙伴之间的高度互动，并将遵循以下主要目标：(A)诱变研究，以确定特定活性位点成分的功能，并扩大可用的产品光谱；(B)重组表达后其他FAE的表征，以研究各自对其他底物的调节，包括各种FAE的结晶，以扩展这些酶的结构知识基础；(C)对激活酶的研究，以建立体外激活。(D)在实验输入的支持下，对FAE反应机制进行多尺度高水平的计算研究，以及(E)开发将FAE及其突变变体应用于新化合物生物技术生产的方法。该项目将采用分子生物学和酶学领域最先进的技术，结合分子建模和生物技术过程的工程。该结果将提高目前对化石燃料和化学工业造成的环境污染的生物修复过程的理解，并将提供基本的生化数据，这将改善微生物在碳氢化合物生物修复中的使用。对整个FAE类的机制的阐明将为这些酶所利用的新的化学反应原理提供新的见解。最后，FAE的研究还将为合成精细化学品和生物可降解聚合物的底物提供新的生物技术工艺工具。

This proposal intends to investigate the biochemistry and reactions mechanisms of a class of novel enzymes which catalyse the radical-based addition of fumarate to a number of highly recalcitrant compounds, like hydrocarbons. These fumarate-adding enzymes (FAE) form a subclass of the glycyl radical enzymes which are highly oxygen-sensitive and need to be activated to the radical state by special activating enzymes. The proposed project is highly interactive between the German and Polish partners and will follow the following major aims: (A) mutagenesis studies to identify the functions of particular active site constituents and to extend the available product spectra, (B) characterization of other FAE after recombinant expression to investigate the respective accommodations to other substrates and including crystallization of various FAE to extend the structural knowledge basis on these enzymes, (C) studies on the activating enzyme to establish in-vitro activation, (D) multiscale high-level computational investigations of the FAE reaction mechanism, backed up with experimental input, and (E) developing methods to apply FAE and their mutant variants for biotechnological production of novel compounds. The project will employ state-of-the-art techniques from the field of molecular biology and enzymology combined with molecular modelling and engineering of biotechnological processes. The results will improve current understanding of bioremediation processes of environmental contaminations caused by fossil fuel and chemical industries and will deliver fundamental biochemical data which will improve the use of microorganisms in bioremediation of hydrocarbons. The elucidation of mechanism for the whole class of FAE will provide new insights into a novel chemical reaction principle exploited by these enzymes. Finally, studies of FAE will also provide new tools of biotechnological processes to synthesized fine chemicals and substrates for biodegradable polymers.