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

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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.