RUI: Biophysical Characterization of Reduced Flavin-Dependent Two Component Monooxygenase Sulfur Assimilation Enzymes: SfnG, MsuC, and MsuD

有了这个奖项，生命过程化学项目将资助萨福克大学的丹尼斯·k·威克特博士和麻省理工大学波士顿分校的丹尼尔·p·道林博士研究从天然有机分子中提取硫的蛋白质。含硫物质的循环对于理解生物体（包括人类）如何在地球的水、大气和陆地组成部分之间移动这种特定元素起着重要作用。含有碳硫键和硫氧键的化合物的相互转化在一系列微生物中被观察到，但化学反应进行的顺序直到最近才被发现。因此，这些化学转化在很大程度上仍未被探索。这项研究需要Wicht博士和Dowling博士共同努力，进行基本的生化分析，并获得这些事件发生的结构信息。本科和研究生阶段的研究助理可以获得酶学、生物化学和蛋白质晶体学方面的经验。此外，参与者还与中学生一起参与外展活动，并获得有关终身学习技能的具体反馈，这些技能对培养现代STEM劳动力至关重要。由PI Wicht和共同PI Dowling提供的强大科学指导有助于培养多元化，具有全球竞争力的STEM劳动力。最近报道的细菌硫同化途径从天然存在的二甲基砜到无机亚硫酸盐是工作的中心焦点。有三种单加氧酶参与生化反应：SfnG（用于砜利用）、MsuC（用于甲磺酸利用）和MsuD（用于甲磺酸利用）。这三种酶都是依赖黄素的还原单加氧酶，需要三种底物：有机硫分子、O2和FMNH2。从二甲基砜到亚硫酸盐的新型生化途径的动力学景观是未知的，因此提出的研究活动的直接目标是利用可见光谱法确定途径中每种蛋白质的表观稳态动力学参数。纯化后的重组酶的动力学分析与1H和13C NMR测定的底物特异性数据相结合。对于每种单加氧酶，定义了酶周转的最大速率和首选有机硫底物的特异性常数。高分辨率x射线晶体学用于确定酶活性位点的分子细节和反应化学。综上所述，我们利用实验结果提出了一个与两个C-S键的断裂和一个S-O键的形成相一致的总体酶机制。绘制这些酶的活性位点环境，通过识别与硫成键和断键有关的重要氨基酸残基，指导未来的机制研究。该奖项反映了美国国家科学基金会的法定使命，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 this award, the Chemistry of Life Processes program is funding Dr. Denyce K. Wicht at Suffolk University and Dr. Daniel P. Dowling at UMass-Boston to research proteins involved in extracting sulfur from naturally occurring organic molecules. The cycling of sulfur-containing substances is important to understanding how living organisms (including humans) play a role in moving this specific element between earth's water, atmosphere, and land components. The interconversions of compounds containing carbon-sulfur bonds and sulfur-oxygen bonds are observed across a range of microorganisms, but the order in which the chemistry proceeds only recently was discovered. Therefore, these chemical transformations remain largely unexplored. This research entails a collaborative effort by Dr. Wicht and Dr. Dowling to perform fundamental biochemical analyses and obtain structural information for how these events take place. Research assistants at the undergraduate and graduate level gain experience with enzymology, biochemistry, and protein crystallography. Additionally, participants engage in outreach activities with middle school students and receive specific feedback on lifelong learning skills important to the production of a modern STEM workforce. Strong scientific mentorship provided by PI Wicht and co-PI Dowling contributes to the development of a diverse, globally competitive STEM workforce. The recently reported bacterial sulfur assimilation pathway from naturally occurring dimethylsulfone to inorganic sulfite is the central focus of the work. There are three monooxygenases involved in the biochemical reactions: SfnG (for sulfone utilization), MsuC (for methanesulfinate utilization), and MsuD (for methanesulfonate ultilization). All three enzymes are reduced flavin dependent monooxygenases that require three substrates: an organosulfur molecule, O2, and FMNH2. The kinetic landscape of the novel biochemical pathway from dimethylsulfone to sulfite is unknown, therefore the immediate goal of the proposed research activities is the determination of the apparent steady-state kinetic parameters for each protein in the pathway using visible spectroscopy. The kinetic analyses of purified recombinant enzymes are coupled with substrate specificity data determined by 1H and 13C NMR spectroscopy. For each monooxygenase, a maximum rate of enzyme turnover and the specificity constant for the preferred organosulfur substrate is defined. High-resolution X-ray crystallography is used to determine the molecular details of enzyme active site(s) and reaction chemistry. Together, the experimental results are used to formulate a proposed overall enzyme mechanism consistent with the cleavage of two C-S bonds and the formation of one S-O bond. Mapping the active site environment of these enzymes guides future mechanistic studies by identifying important amino acid residues involved in bond-making and bond-breaking to sulfur.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.