Collaborative Research: Ethylene-Forming Enzyme

在化学系生命过程化学项目的支持下，密歇根州立大学的Robert Hausinger和Jian Hu以及密歇根理工大学的Christo Christov正在研究乙烯形成酶（EFE）的催化策略。这种细菌或真菌蛋白是非血红素Fe（II）和2-酮戊二酸（2 OG）依赖性加氧酶的成员，并且不同于也形成乙烯但通过不同过程的众所周知的植物酶。EFE催化两种不同的反应，一种产生乙烯，另一种产生富氮化合物胍。乙烯是一种天然的植物催熟激素，已被作为汽油的替代燃料。 胍是一种很有潜力的氮肥。因此，生产乙烯和胍的反应具有根本意义，并具有潜在的国家经济效益和社会影响。EFE酶的研究将为两名博士后科学家和本科生提供生物化学，结构和计算方法的高级培训。该项目取得的科学进展将纳入大学研究生课程。此外，这些研究的结果将通过在大学科学节上的演讲传达给终身学习者。该建议的总体目标是阐明EFE的催化机制相结合的实验和计算研究。该过程的关键是研究酶的两个反应：（1）2 OG分解为三个分子的二氧化碳/碳酸氢盐和乙烯，以及（2）2 OG转化为琥珀酸盐和二氧化碳，并将氨基酸L-精氨酸（L-Arg）转化为胍和L-Δ1-吡咯啉-5-羧酸盐。为了了解控制这两个反应的相对活性的分子决定因素，生物化学，结构和计算方法将被应用于最好的研究EFE的定点变异体从细菌假单胞菌，EFE同系物从真菌指状青霉，并重建祖先EFE蛋白。利用这些信息，沿着结构导向蛋白质工程，将创建优化用于生产生物燃料乙烯或植物肥料胍的EFE变体。这项工作显着扩展了其他重要的Fe（II）和2 OG依赖的加氧酶，不能催化这些reactions.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持结构/机制的信息。

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Robert Hausinger and Jian Hu at Michigan State University and Christo Christov at Michigan Technological University are studying the catalytic strategy of the ethylene-forming enzyme (EFE). This bacterial or fungal protein is a member of the non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases and is distinct from the well-known plant enzyme that also forms ethylene but by a distinct process. EFE catalyzes two distinct reactions, with one producing ethylene and the other generating the nitrogen-rich compound guanidine. Ethylene is the natural plant-ripening hormone and has been advanced as a fuel source alternative to gasoline. Guanidine is a potential nitrogen fertilizer. Thus, the reactions that produce ethylene and guanidine are of fundamental interest and have potential national economic benefits and societal impacts. Investigation of the EFE enzyme will provide advanced training in biochemical, structural, and computational approaches to two postdoctoral scientists and to undergraduate students. Scientific advances obtained from this project will be incorporated into university graduate courses. In addition, the findings from these studies will be communicated to lifelong learners in the general public via presentations at the university Science Festival. The overarching goal of this proposal is to elucidate the catalytic mechanism of EFE by a combination of experimental and computational studies. Critical to this process are studies that examine the enzyme’s two reactions: (1) decomposition of 2OG into three molecules of carbon dioxide/bicarbonate and ethylene and (2) the conversion of 2OG to succinate and carbon dioxide and transformation of the amino acid L-arginine (L-Arg) into guanidine and L-Δ1-pyrroline-5-carboxylate. To understand the molecular determinants that control the relative activities for the two reactions, biochemical, structural, and computational approaches will be applied to site-directed variants of the best studied EFE from the bacterium Pseudomonas syringae, an EFE homolog from the fungus Penicillium digitatum, and to reconstructed ancestral EFE proteins. Using this information along with structure-guided protein engineering, EFE variants that are optimized for producing the biofuel ethylene or the plant fertilizer guanidine will be created. This work significantly extends the structural/mechanistic information available for other important Fe(II)- and 2OG-dependent oxygenases that are unable to catalyze these reactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.