RUI: Re-engineering Ring-Cleaving Dioxygenases for Activity Towards Novel Substrates

RUI：重新设计开环双加氧酶以实现新型底物的活性

• 批准号: 2203928
• 负责人: Timothy Machonkin
• 金额: $ 24万
• 依托单位: Whitman College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203928&HistoricalAwards=false
• 关键词: RUI; Re; engineering; Ring; Cleaving

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Timothy Machonkin from Whitman College is studying a class of deoxygenate enzymes found in soil bacteria that are capable of oxidatively breaking down certain toxic organic compounds that are common environmental pollutants. A key step in this process is breaking open the six-carbon benzene ring in these compounds, which is performed by the ring-cleaving dioxygenases that are the topic of this proposals. Two of these ring-cleaving dioxygenases are less studied but are attractive targets for enzyme reengineering efforts with the aim of finding more active variants with respect to chlorinated aromatic organic compounds. Since such chlorinated compounds are among the most intractable pollutants, utilizing enzymes to break down these compounds could be of use in bioremediation efforts. This project will support summer research experiences for Whitman undergraduate students, immersing them in modern bioinorganic chemistry research and providing them with opportunities to present and publish their research. This work is also expected to contribute to pedagogical improvement in the chemistry curriculum and enhance community science outreach efforts at Whitman College. This work will focus examining both a hydroquinone dioxygenase and an aminophenol dioxygenase enzyme in order to establish structure/activity profiles of these enzymes across a range of mutants and a set of targeted substrates. These enzymes provide an ideal platform for enzyme engineering because they are structurally characterized, easily expressed, highly active, and already fairly tolerant of different substrates. Site-directed mutagenesis, particularly targeting removing specific bulky active site amino acids will be performed to test whether this serves to broaden substrate specificity, specifically with a focus on chlorinated aromatic substrates. Steady-state kinetic characterization will be performed to define how these mutations have affected the enzyme activity, which should assist in mapping out determinants of substrate specificity. In addition, computational studies will be used to better understand the fundamental reasons why chlorinated substrates remain difficult targets for this class of enzymes. Success at generating mutant enzymes with improved activity toward substrates with different patterns of chloro-substitution has the potential to provide insight into how these enzymes bind chlorinated substrates as well as guide future efforts to engineer ring-cleaving dioxygenase enzymes.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.

在化学系生命过程化学（CLP）项目的支持下，惠特曼学院的 Timothy Machonkin 正在研究土壤细菌中发现的一类脱氧酶，这些酶能够氧化分解某些常见环境污染物的有毒有机化合物。该过程的关键步骤是打开这些化合物中的六碳苯环，这是由本提案主题的开环双加氧酶完成的。其中两种断环双加氧酶研究较少，但对于酶再工程工作来说是有吸引力的目标，目的是寻找对于氯化芳香族有机化合物更具活性的变体。由于此类氯化化合物是最难处理的污染物之一，因此利用酶分解这些化合物可用于生物修复工作。该项目将为惠特曼本科生提供暑期研究体验，让他们沉浸在现代生物无机化学研究中，并为他们提供展示和发表研究成果的机会。这项工作预计还将有助于化学课程的教学改进，并加强惠特曼学院的社区科学推广工作。这项工作将重点检查对苯二酚双加氧酶和氨基酚双加氧酶，以便在一系列突变体和一组目标底物中建立这些酶的结构/活性概况。 这些酶为酶工程提供了理想的平台，因为它们具有结构特征、易于表达、高活性，并且对不同底物具有相当的耐受性。将进行定点诱变，特别是针对去除特定的大体积活性位点氨基酸，以测试这是否有助于扩大底物特异性，特别是重点关注氯化芳香族底物。将进行稳态动力学表征，以确定这些突变如何影响酶活性，这应有助于找出底物特异性的决定因素。此外，计算研究将用于更好地理解氯化底物仍然是此类酶的困难目标的根本原因。成功生成对具有不同氯取代模式的底物具有更高活性的突变酶有可能深入了解这些酶如何结合氯化底物，并指导未来设计开环双加氧酶的工作。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查进行评估，被认为值得支持 标准。