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

在化学系生命过程化学（CLP）项目的支持下，来自惠特曼学院的蒂莫西·马琼金正在研究一类在土壤细菌中发现的脱氢酶，这些细菌能够氧化分解某些有毒的有机化合物，这些有机化合物是常见的环境污染物。这一过程的关键步骤是打破这些化合物中的六碳苯环，这是由环裂解双加氧酶完成的，这是本提案的主题。这些环裂解双加氧酶的研究较少，但有吸引力的目标，酶的再造努力，目的是找到更多的活性变体，相对于氯代芳香族有机化合物。由于这种氯化化合物是最难处理的污染物之一，利用酶来分解这些化合物可能在生物修复工作中有用。该项目将支持惠特曼本科生的夏季研究经验，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。这项工作也有望有助于化学课程教学的改进，并加强社区科学在惠特曼学院的推广工作。这项工作将集中检查两个氢醌双加氧酶和氨基酚双加氧酶，以建立这些酶的结构/活性分布在一系列的突变体和一组有针对性的基板。 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，并且已经相当耐受不同的底物。将进行定点诱变，特别是靶向去除特定的大体积活性位点氨基酸，以检测这是否有助于拓宽底物特异性，特别是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，这将有助于绘制出底物特异性的决定因素。此外，计算研究将被用来更好地了解氯化底物仍然是这类酶的困难目标的根本原因。成功地产生对不同氯取代模式的底物具有改进活性的突变酶，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知识价值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 (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.