Structures and Mechanisms of Two Enzymes that Produce Hydrocarbon Fuels from Abundant Metabolites

宾夕法尼亚州立大学化学系的“生命过程化学”项目正在支持宾夕法尼亚州立大学研究两种酶的工作，这两种酶催化脂肪酸（丰富和常见的细胞代谢物）转化为碳氢化合物（烷烃和烯烃）的关键步骤。然后，它们可以用作内燃机的插入式生物燃料。这项得到支持的研究正在影响可再生能源的发展，因为利用微生物生产生物燃料是一种很有前途的策略。这个过程特别环保，因为它最终是碳中和的，由太阳能驱动。该项目正在研究上述酶如何发挥作用，并着眼于将它们导入可以生产生物燃料的基因工程微生物中。该奖项支持的工作还为学生和博士后学者提供了在生物物理方法方面接受广泛培训的机会。它还支持两年一次的研讨会，由宾夕法尼亚州立大学金属生物化学家领导，并得到其他机构同事的支持，培训学生和博士后学者在该研究领域使用的独特方法。基因上可操纵的微生物可以(1)从阳光和二氧化碳中产生脂肪酸（直接通过使用光合作用的蓝藻细菌，或间接通过给工程微生物喂食从快速生长和高产的植物（如甘蔗）中获得的糖），(2)将脂肪酸转化为“插入式”生物燃料，作为可再生燃料生物生成的载体被积极追求。最近发现的两种将脂肪酸转化为直链烷烃或烯烃的酶途径涉及在其活性位点含有含铁辅助因子的酶。第一个例子涉及蓝细菌中的两步烷烃生物合成途径，通过脂肪醛将脂肪酸转化为烷烃（a/e）。该途径的第二步由醛去甲酰基加氧酶（ADO）催化，ADO是类铁蛋白二铁羧酸氧化酶和加氧酶的一员。在之前的资助周期中，我们将ADO反应作为一种新型的隐式氧化还原氧化反应进行了研究。进一步的机理研究揭示了潜在的低效率和脆弱性，这可能会限制这种途径在生物燃料生产中的实用性。我们建议对ADO的反应进行更深入的研究，期望进一步的机理认识将指导生物燃料生产微生物的合理设计。第二个例子涉及最近发现的铁酶UndA，它利用氧气将脂肪酸脱羧为二氧化碳和末端烯烃。我们的初步工作表明，UndA也是一种双铁酶，与已发表的假设相反，它是一种单核铁酶。我们建议确定正确的辅因子，并确定详细的分子机制。这些研究将对正在进行的旨在利用这种代谢途径生产生物燃料的研究工作作出重要贡献。

With this award the "Chemistry of Life Processes" program of the "Division of Chemistry" is supporting work at the Pennsylvania State University to study two enzymes that catalyze key steps in the conversion of fatty acids (abundant and common cellular metabolites) to hydrocarbons (alkanes and alkenes). These can then be used as drop-in biofuels for combustion engines. The research supported is impacting development of renewable energy, because the use of microorganisms is a promising strategy for biofuel production. This process is particularly environmentally friendly, because it is ultimately carbon neutral and driven by solar energy. The project is studying how the enzymes described above function with an eye to importing them into genetically engineered microorganisms which can produce biofuels. Work supported by this award is also offering students and postdoctoral scholars opportunities to be broadly trained in biophysical methods. It also supports a biennial workshop, led by Penn State metallobiochemists and supported by colleagues from other institutions, that trains students and postdoctoral scholars in unique methods used in this research field. Genetically manipulable microorganisms that could (i) produce fatty acids from sunlight and carbon dioxide (either directly by using photosynthetic cyanobacteria, or indirectly by feeding the engineered microorganisms sugars obtained from fast-growing and high-yielding plants such as sugar cane) and (ii) convert the fatty acids into "drop-in" biofuels are actively pursued as vehicles for biogenesis of renewable fuels. Two recently discovered enzyme pathways for the conversion of fatty acids into linear alkane or alkene hydrocarbons involve enzymes that harbor iron-containing cofactors at their active site. The first example involves the two-step alkane biosynthesis pathway in cyanobacteria that converts fatty acids via fatty aldehydes to alk(a/e)nes. The second step in this pathway is catalyzed by the enzyme aldehyde-deformylating oxygenase (ADO), a member of the ferritin-like diiron-carboxylate oxidases and oxygenases. In the previous funding cycle we studied the ADO reaction as a novel, cryptically redox oxygenation reaction. Further mechanistic studies revealed potential inefficiencies and vulnerabilities that could limit the usefulness of this pathway for biofuel production. We propose to study the reaction of ADO in greater depth, with the expectation that further mechanistic insight will guide the rational design of biofuel-producing microorganisms. The second example involves a recently discovered iron-enzyme, UndA, that utilizes O2 to decarboxylate fatty acids to carbon dioxide and terminal alkenes. Our preliminary work suggests that UndA is also a diiron enzyme, contrary to the published assumption that it is a mononuclear iron enzyme. We propose to identify the correct cofactor and determine the molecular mechanism in detail. These studies will be an important contribution to ongoing research efforts that aim at utilizing this metabolic pathway for biofuel production.