With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Michael McLeish from Indiana University-Purdue University Indianapolis to study how enzymes evolve to choose their substrate and products. Many enzymes use thiamin, in the form of thiamin diphosphate, as a cofactor to help them speed up biological reactions. Several of these enzymes use pyruvate as a primary substrate, but have evolved to convert it to different products. This project will use directed evolution to generate new thiamin enzymes that use pyruvate. A combination of steady-state kinetics and 1H NMR and CD spectroscopy will be used to determine rate constants for the individual chemical steps in the reactions of these new enzymes and X-ray crystallography will be used to determine the structures of the evolved enzymes. The kinetics of the new enzymes will be compared to those of their naturally-occurring counterparts and related to any structural differences between these two types of enzymes. The goal of the project is to attain a fundamental understanding of the principles guiding the evolution of substrate and reaction specificity.
Most enzymes are very specialized and have evolved to recognize only one or two reactants and form one or two products. This project will explore the way in which an unusual group of enzymes is able to operate in the same manner using the same reactant, but turn the reactant into different products. Understanding this process is significant because this group of enzymes can be used in industrially important reactions. The study will require the evolution of enzymes with altered substrate specificity, and will allow undergraduate and graduate students an opportunity to explore both chemical and biological processes. They will receive training in microbiology, molecular biology, enzyme kinetics, NMR spectroscopy and X-ray crystallography. Importantly, they will also be able to integrate these studies with an outreach component that includes developing a teaching unit and a hands-on enzymology project for middle school students.
