CAREER: Next Generation Kinetic Isotope Effect Measurements for the Analysis of Glycosyltransferase Enzyme Mechanisms

职业：用于糖基转移酶机制分析的下一代动力学同位素效应测量

• 批准号: 1945162
• 负责人: Myles Poulin
• 金额: $ 69万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1945162&HistoricalAwards=false
• 关键词: CAREER; Next; Generation; Kinetic; Isotope

Glycosyltransferases are enzymes that chemically facilitate (catalyze) the assembly of sugars into complex long chain polymers. Such polymers are involved in a number of different biological processes, including formation of biofilms that protect microbes at surfaces (such as the surfaces of teeth) and pathways that lead to antibiotic resistance. With this CAREER award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Myles Poulin of the University of Maryland at College Park to develop new methods to study the transient chemical structures (the transition states) that are key to the reactions in the catalytic mechanisms of bacterial glycosyltransferase enzymes. Dr. Poulin is developing a new quantitative approach to precisely follow how heavier atoms affect the reactions along catalytic pathways (kinetic isotope effects). Combining these precise measurements with computational studies allows for detailed analyses of transition state structures in the mechanisms of glycosyltransferase enzymes. The results of this research help in the design of new antimicrobial agents that target biofilm formation and antibiotic resistance pathways. In addition to these research activities, Dr. Poulin is developing an integrated educational outreach program that engages high school and first year university students in research experiences and hands-on laboratory activities. The goal of this program is to increase enrollment and retention of underrepresented minority students in science, technology, engineering, and mathematics fields.Glycosyltransferase enzymes play a critical role in numerous biological processes and yet, the transition states for these enzyme reactions have not been studied in detail. Kinetic isotope effect (KIE) measurements are a powerful tool to study enzyme mechanisms and transition states, which further help guide the design of potent transition state analogues as inhibitors. However, KIE measurements are not widely applied due to the complex analytical workflows required to measure KIEs with high precision. The primary objective of this research is to develop a general, quantitative, whole molecule mass spectrometry approach for KIE determination that simplifies the analytical workflow required to precisely measure heavy atom KIEs. This approach is applied to study the mechanisms and transition state structures for two bacterial glycosyltransferase enzymes: PgaCD, which is involved in bacterial biofilm exopolysaccharide assembly, and BshA, which functions in Fosfomycin antibiotic resistance. Combining these highly precise KIE measurements with computational modeling provides a detailed picture of the transition states of the enzymes. This research may provide important new insights into the mechanisms of glycosyltransferase enzymes involved in the assembly of exopolysaccharide biofilms and the biosynthesis of bacillithiol. Results from these studies may also help guide the design and development of inhibitors targeting these pathways. In addition, the methods developed from this project are generally applicable to other enzymes and are of general interest to researchers studying enzyme mechanisms and thus this research topic is included in NSF's Understanding the Rules of Life Big Idea.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.

糖基转移酶是化学促进（催化）糖组装成复杂长链聚合物的酶。这些聚合物参与了许多不同的生物过程，包括形成生物膜，保护表面（如牙齿表面）的微生物和导致抗生素耐药性的途径。有了这个CAREER奖，化学部的生命过程化学计划正在资助马里兰州大学帕克分校的Myles Poulin博士开发新方法来研究瞬态化学结构（过渡态），这是细菌糖基转移酶催化机制中反应的关键。Poulin博士正在开发一种新的定量方法，以精确跟踪重原子如何影响沿着催化途径的反应（动力学同位素效应）。将这些精确的测量与计算研究相结合，可以详细分析糖基转移酶机制中的过渡态结构。这项研究的结果有助于设计针对生物膜形成和抗生素耐药性途径的新型抗菌剂。除了这些研究活动，Poulin博士正在开发一个综合的教育推广计划，让高中和大学一年级的学生参与研究经验和动手实验室活动。该计划的目标是增加在科学，技术，工程和数学领域中代表性不足的少数民族学生的入学率和保留率。糖基转移酶在许多生物过程中起着关键作用，然而，这些酶反应的过渡态还没有被详细研究。动力学同位素效应（KIE）测量是研究酶作用机制和过渡态的有力工具，有助于指导有效的过渡态类似物作为抑制剂的设计。然而，KIE测量没有被广泛应用，由于需要复杂的分析工作流程来测量KIE具有高精度。本研究的主要目的是开发一种通用的，定量的，全分子质谱法测定KIE，简化了精确测量重原子KIE所需的分析工作流程。这种方法适用于研究两种细菌糖基转移酶的机制和过渡态结构：PgaCD，这是参与细菌生物膜胞外多糖组装，和BshA，这在磷霉素抗生素耐药性的功能。将这些高精度的KIE测量与计算建模相结合，提供了酶过渡态的详细图像。本研究为糖基转移酶参与胞外多糖生物膜的组装和细菌硫醇的生物合成提供了重要的新见解。这些研究的结果也可能有助于指导针对这些途径的抑制剂的设计和开发。此外，该项目开发的方法普遍适用于其他酶，也是研究酶机制的研究人员普遍感兴趣的研究课题，因此该研究课题被列入NSF的Understanding the Rules of Life Big Idea。该奖项反映了NSF的法定使命，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。