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

RUI：减少黄素依赖性双组分单加氧酶硫同化酶的生物物理表征：SfnG、MsuC 和 MsuD

• 批准号: 1807480
• 负责人: Denyce Wicht
• 金额: $ 29.4万
• 依托单位: Suffolk University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1807480&HistoricalAwards=false
• 关键词: RUI; Biophysical; Characterization; Reduced; Flavin

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.

有了这个奖项，生命过程的化学计划是资助博士Denyce K。萨福克大学的Wicht和麻省大学波士顿分校的丹尼尔P. Dowling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，大气和陆地成分之间移动这种特定元素中发挥作用非常重要。在一系列微生物中观察到含有碳-硫键和硫-氧键的化合物的相互转化，但化学过程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博士的合作努力，以进行基本的生化分析，并获得这些事件如何发生的结构信息。本科生和研究生水平的研究助理获得酶学，生物化学和蛋白质晶体学的经验。此外，参与者与中学生一起参加外联活动，并获得有关终身学习技能的具体反馈，这些技能对现代STEM劳动力的生产至关重要。PI Wicht和共同PI Dowling提供的强大的科学指导有助于发展多元化，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无机亚硫酸盐是工作的中心焦点。有三种单加氧酶参与生物化学反应：SfnG（用于砜利用）、MsuC（用于甲亚磺酸盐利用）和MsuD（用于甲磺酸盐利用）。所有这三种酶都是还原型黄素依赖性单加氧酶，需要三种底物：有机硫分子、O2和FMNH 2。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，拟议的研究活动的直接目标是使用可见光谱法测定途径中每种蛋白质的表观稳态动力学参数。纯化的重组酶的动力学分析与通过1H和13 C NMR光谱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优选的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和反应化学的分子细节。总之，实验结果被用来制定一个建议的整体酶的机制与两个C-S键的裂解和一个S-O键的形成一致。绘制这些酶的活性位点环境，通过识别与硫键形成和键断裂有关的重要氨基酸残基，指导未来的机理研究。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。

项目成果

Structure and function of the two-component flavin-dependent methanesulfinate monooxygenase within bacterial sulfur assimilation

细菌硫同化中双组分黄素依赖性甲亚磺酸单加氧酶的结构和功能

• DOI: 10.1016/j.bbrc.2019.11.008
• 发表时间: 2020
• 期刊: Biochemical and Biophysical Research Communications
• 影响因子: 3.1
• 作者: Soule, Jess;Gnann, Andrew D.;Gonzalez, Reyaz;Parker, Mackenzie J.;McKenna, Kylie C.;Nguyen, Son V.;Phan, Ngan T.;Wicht, Denyce K.;Dowling, Daniel P.
• 通讯作者: Dowling, Daniel P.