Monooxygenase/arylamine N-oxygenase activity within a single non-heme diiron enzyme (MiaE)

得克萨斯大学阿灵顿分校的布拉德·S·皮尔斯博士将利用美国国家科学基金会化学部门生命过程化学项目授予的这一奖项，研究影响生物氧化程度的因素。拟议的活动为更好地了解驱动生物氧化的结构因素提供了机会，从而为合理设计生物启发或生物工程氧化催化剂提供了一个框架。氧化作用导致电子从初始分子(或底物)向分子氧的净转移。从会计的角度来看，氧化的“程度”可以定义为转移的电子的数量。以前已经注意到，铁周围结构的变化极大地影响了催化剂对底物的氧化程度。拟议的生物化学研究利用铁催化剂在一个简化的单一平台上比较指导2-电子和6-电子氧化的因素。除了对研究生/本科生进行生物化学、生物物理学和化学催化方面的培训外，该奖项的更广泛的影响活动还包括为PI和参与国家科学基金会资助的活动的学生(本科生和研究生)提供科学交流方面的培训和实践经验。非血红素双铁酶是一个普遍存在的酶家族，能够催化各种令人惊叹的生物氧化反应。在这些酶中，已经观察到第一铁配位球内单一氨基酸的扰动对O2-激活后产生的瞬时中间产物的性质和底物氧化的程度有深远的影响。例如，在细菌多组分单加氧酶(BMM)超家族中，羟基酶组分二铁位点由2-组氨酸和4-羧酸(天冬氨酸或谷氨酸)残基[2-组氨酸/4-羧酸]配位。这些酶的还原(二亚铁)活性中心能够还原激活分子氧，以催化其特定底物的2电子氧化。相比之下，已鉴定出少数酶含有一个额外的组氨酸残基，配位到两个铁位之一；导致[3-组氨酸/4-羧酸盐]二铁簇。其中包括芳胺N-加氧酶类的非血红素双铁酶AurF和CmlI。这些酶催化芳胺底物发生显着的6电子氧化反应，生成硝基芳基产物。建议的研究利用从鼠伤寒沙门氏菌(St Miae)分离的一种不寻常的非血红素tRNA羟基酶(St Miae)来比较指导(6电子)芳胺N加氧酶和(2电子)单加氧酶化学的相关因素在单一的酶平台上。这种比较是通过ST MIAE活性中心内的单一氨基酸取代(L199H)实现的，该取代产生[3-His/4-羧酸盐]第一铁配位球并赋予6-电子芳香胺N-加氧酶活性。除了对研究生/本科生进行生物化学、生物物理学和化学催化方面的培训外，该奖项的更广泛的影响活动还包括为PI和参与国家科学基金会资助的活动的学生(本科生和研究生)提供科学交流方面的培训和实践经验。

With this award granted by the National Science Foundation-Chemistry Division, Chemistry of Life Processes Program, Dr. Brad S. Pierce at The University of Texas at Arlington will investigate the factors influencing the extent of biological oxidations. The proposed activities provide an opportunity to better understand structural factors driving biological oxidations and thus provide a framework for the rational design of biologically-inspired or bioengineered oxidation catalysts. Oxidations result in a net transfer of electrons from the initial molecule (or substrate) to molecular oxygen. From an accounting point of view, the "extent" of an oxidation can be defined as the number of electrons transferred. It has been previously noted that changes in structure around iron dramatically influence the extent of substrate oxidation by catalysts. The proposed biochemical studies utilize an iron catalyst to compare the factors directing 2- and 6-electron oxidations within a single, simplified platform. Beyond training for graduate/undergraduate students in biochemistry, biophysics and chemical catalysis, broader impact activities for this award include training and practical experience in scientific communication for the PI and students (undergraduate and graduate) participating in NSF-supported activities. Non-heme diiron enzymes are a ubiquitous family of enzymes capable of catalyzing an amazing diversity of biological oxidations. Among these enzymes, it has been observed that single amino acid perturbations within the first Fe-coordination sphere have a profound impact on the nature of transient intermediates produced following O2-activation and the extent of substrate oxidation. For example, within the bacterial multicomponent monooxygenase (BMM) superfamily, the hydroxylase component diiron site is coordinated by 2-histidine and 4-carboxylate (Asp or Glu) residues [2-His/4-carboxylate]. The reduced (diferrous) active sites of these enzymes are capable of reductively activating molecular oxygen to catalyze the 2-electron oxidation of its specific substrate. By contrast, a small number of enzymes have been identified which contain an additional His-residue coordinated to one of the two Fe-sites; resulting in a [3-His/4-carboxlate] diiron cluster. Among these are the arylamine N-oxygenase class of non-heme diiron enzymes AurF and CmlI. These enzymes catalyze a remarkable 6-electron oxidation of arylamine substrates to yield a nitroaryl product. The proposed studies utilize an unusual non-heme tRNA-hydroxylase isolated from S. typhimurium (St MiaE) to compare the relevant factors directing (6-electron) arylamine N-oxygenase and (2-electron) monooxygenase chemistry within a single, enzymatic platform. This comparison is made possible by a single amino acid substitution (L199H) within the St MiaE active site which yields a [3-His/4-carboxylate] first Fe-coordination sphere and imparts 6-electron arylamine N-oxygenase activity. Beyond training for graduate/undergraduate students in biochemistry, biophysics and chemical catalysis, broader impact activities for this award include training and practical experience in scientific communication for the PI and students (undergraduate and graduate) participating in NSF-supported activities.