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铁(II)和2-氧戊二酸(2OG)依赖的加氧酶的成员，不同于众所周知的植物酶，后者也形成乙烯，但通过不同的过程。EFe催化两个不同的反应，一个产生乙烯，另一个产生富氮化合物胍。乙烯是一种天然的植物成熟激素，已被用作汽油的替代燃料。胍是一种很有潜力的氮肥。因此，产生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。对EFE酶的研究将为两名博士后科学家和本科生提供生化、结构和计算方法方面的高级培训。从这个项目中获得的科学进步将被纳入大学研究生课程。此外，这些研究的发现将通过在大学科学节上的演讲向普通公众的终身学习者传达。该方案的首要目标是通过实验和计算相结合的研究来阐明EFE的催化机理。这一过程的关键是研究该酶的两个反应：(1)将2OG分解成二氧化碳/重碳酸盐和乙烯三个分子，(2)将2OG转化为琥珀酸和二氧化碳，以及将氨基酸L-精氨酸(L-精氨酸)转化为胍和L-Δ1-吡咯烷-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，生化、结构和计算方法将被应用于研究最深入的丁香假单胞菌EFE的定点变体，以及重建祖先EFE蛋白。利用这些信息和结构引导的蛋白质工程，将创造出优化用于生产生物燃料乙烯或植物肥料胍的EFE变体。这项工作大大扩展了其他重要的依赖Fe(II)和2OG的加氧酶的结构/机制信息，这些加氧酶无法催化这些反应。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.