CAREER:Investigating Mechanisms of Regulatory and Functional Diversity in an Enzyme Superfamily

University Of Alabama Tuscaloosa, Tuscaloosa AL

Investigators

Abstract

Intellectual Merit: Allosteric regulation is a central tenet of modern biochemistry, however, it is a mechanism that is still not well understood. The long-term goal of this project is to understand how allosteric and catalytic mechanisms work together in multi-domain enzymes. This research examines the mechanistic and evolutionary interactions between two conserved domains in the context of the DRE-TIM metallolyase superfamily. An integrated experimental approach, including biochemical, structural, and bioinformatics techniques is used to (a) determine the identity of kinetic steps perturbed in allosteric inhibition, (b) predict and verify functional and structural diversity and (c) investigate the role of conserved residues in the allosteric mechanism. This type of approach overcomes several difficulties commonly encountered in the study of allosteric enzymes, moves away from the phenomenological description of different allosteric mechanisms and considers the impact of evolutionary pressures on the diversity of mechanisms within a conserved protein scaffold. Rapid-reaction kinetics and kinetic isotope effects will be used to investigate the changes to enzyme mechanisms caused by allosteric inhibitors. The creation of a protein similarity network for the model allosteric domain will provide rapid organization for a large number of diverse sequences predicted to contain the domain. High-throughput structural techniques will provide essential structural coverage for clusters of sequences currently lacking structural description. Mechanistic and structural data will be mapped onto the protein similarity network to define the functional and structural boundaries of the model allosteric domain. The integrated results will provide a description of structure/function relationships contributing to functional and allosteric diversity and also give insight into how a single protein scaffold evolves multiple allosteric mechanisms. The outcomes of this research will impact the growing areas of allosteric biosensors and metabolic engineering of bacterial strains for industrial uses. Broader Impacts: This project will promote interdisciplinary training of graduate and undergraduate students at The University of Alabama. Graduate student training will be enhanced by an on-site visit to the laboratory of Dr. Patricia Babbitt at the University of California, San Francisco for hands-on training in cutting edge bioinformatics techniques. Undergraduate training will be accomplished through two mechanisms designed to increase exposure to original hypothesis-based research. The first mechanism will provide multiple positions in the Frantom laboratory for undergraduate researchers from the University of Alabama Emerging Scholars to continue performing mentored research. The second mechanism is the creation of a formal undergraduate laboratory course to mimic key experiences encountered in undergraduate research. Students unable to participate in direct scientific research will benefit from a laboratory course built around performing original hypothesis-driven experiments that contribute directly to the intellectual merit of this project.