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.
