Excellence in Research: Elucidating Factors that Affect Substrate Promiscuity of Bacterial Capsule Polymerases for Synthesis of Novel Polysaccharides

非技术摘要：水中重金属污染造成环境风险。细菌碳水化合物生物材料由于其化学性质，可以很好地与金属阳离子结合，因此在开发去除环境重金属污染的技术等方面具有潜力。尽管是主要的大分子之一，但这种细菌碳水化合物生物材料以及合成它们的酶还没有完全被了解。更具体地说，对(1)大多数碳水化合物产生酶的三维结构和(2)确定这些酶可以使用哪些特定类型的化合物来合成新的碳水化合物生物材料结构的规则缺乏了解。这个项目的研究团队试图利用计算和实验室研究方法获得这两个领域的基础知识。这项工作的结果将为未来的研究提供重要的发现，从而设计具有增强的金属结合性能的碳水化合物生物材料。拟议的工作还将通过摩根州立大学的研究机会和课程修订，扩大代表不足的少数群体(URM)对生物材料研究和先进计算工具的参与。技术摘要：在许多革兰氏阴性菌的外表面发现的细菌被膜多糖具有不同的长度、组成和结构性质。这些聚合物可能为生物修复、货物输送系统和生物活性聚合物提供新的环境友好的生物材料来源。了解产生胶囊的酶对于设计新的碳水化合物结构以服务于这些应用是至关重要的。然而，这些酶中的许多都被研究得很少，而且很少有已知的三维结构。该项目旨在研究脑膜炎奈瑟氏菌W群(NMW)胶囊聚合酶核苷酸供体底物专一性的规则，以此为基础研究其作为合成新多糖结构的酶工具的潜力。其长期目标是设计具有改善金属修复活性的多糖基生物材料。项目组将协调工作，以实现以下目标：(1)使用建模、分子动力学模拟和突变方法确定调节NMW胶囊聚合酶特异性的关键氨基酸；(2)使用表面等离子体共振和生物发光分析确定天然和修饰底物的结合动力学和酶活性；以及(3)对拟议的新型碳水化合物聚合物进行计算建模和分子动力学模拟，并表征酶产生的多糖。拟议的工作将通过高中、本科生和研究生的学生研究机会，扩大未被充分代表的少数群体(URM)对生物材料研究和先进计算工具的参与。此外，基于研究的生物信息学活动将被纳入本科生和研究生的生物化学课程。因此，该项目将在STEM工作队伍中为来自URM的学生提供研究和教育培训。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical Abstract: Heavy metal pollution of water poses environmental risks. Bacterial Carbohydrate biomaterials, due to their chemical properties, can bind to metal cations very well and therefore have the potential to be used in the development of technologies for removing environmental heavy metal contamination, among other applications. Despite being one of the major classes of macromolecules, such bacterial carbohydrate biomaterials as well as the enzymes that synthesize them are not fully understood. More specifically, there is a lack of knowledge of (1) the three-dimensional structure of most carbohydrate-producing enzymes and (2) the rules that determine what specific types of compounds these enzymes could use to synthesize new carbohydrate biomaterial structures. The team of researchers on this project seek to gain fundamental knowledge into these two areas using computational and laboratory research approaches. Outcomes of this work will provide key findings to inform future studies leading to the design of carbohydrate-based biomaterials with enhanced metal-binding properties. The proposed work will also broaden participation of underrepresented minorities (URMs) in biomaterials research and advanced computational tools through research opportunities and curricular revisions at Morgan State University. Technical Abstract: Bacterial capsular polysaccharides, found on the outer surface of many Gram-negative bacteria, differ in length, composition, and structural properties. These polymers may provide new environmentally friendly sources of biomaterials for bioremediation, cargo-delivery systems, and biologically active polymers. An understanding of capsule-producing enzymes is critical to the design of new carbohydrate structures to serve in these applications. However, many of these enzymes have been poorly studied and there are very few known 3-dimensional structures. The proposed project seeks to study the rules governing nucleotide donor substrate specificity of the Neisseria meningitidis serogroup W (NmW) capsule polymerase as a basis for investigating its potential to serve as an enzymatic tool for the synthesis of novel polysaccharide structures. The long-term goal is to design polysaccharide-based biomaterials with improved metal remediation activity. The project team will work in coordination to carry out the following objectives: (1) Identification of key amino acids that modulate specificity of the NmW capsule polymerase using modeling, molecular dynamics simulations and mutational approaches; (2) Determination of binding kinetics and enzymatic activity of natural and modified substrates using surface plasmon resonance and bioluminescence assays; and (3) Computational modeling and molecular dynamics simulations of proposed novel carbohydrate polymers and characterization of enzymatically-produced polysaccharides. The proposed work will broaden participation of underrepresented minorities (URMs) in biomaterials research and advanced computational tools through student research opportunities at the high school, undergraduate and graduate level. In addition, a research-based bioinformatics activity will be incorporated into undergraduate and graduate Biochemistry courses. Thus, this project will provide research and educational training to students from URMs in the STEM workforce.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.