RUI: Re-engineering Ring-Cleaving Dioxygenases for Activity Towards Novel Substrates

Whitman College, Walla Walla WA

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Timothy Machonkin from Whitman College is studying a class of deoxygenate enzymes found in soil bacteria that are capable of oxidatively breaking down certain toxic organic compounds that are common environmental pollutants. A key step in this process is breaking open the six-carbon benzene ring in these compounds, which is performed by the ring-cleaving dioxygenases that are the topic of this proposals. Two of these ring-cleaving dioxygenases are less studied but are attractive targets for enzyme reengineering efforts with the aim of finding more active variants with respect to chlorinated aromatic organic compounds. Since such chlorinated compounds are among the most intractable pollutants, utilizing enzymes to break down these compounds could be of use in bioremediation efforts. This project will support summer research experiences for Whitman undergraduate students, immersing them in modern bioinorganic chemistry research and providing them with opportunities to present and publish their research. This work is also expected to contribute to pedagogical improvement in the chemistry curriculum and enhance community science outreach efforts at Whitman College. This work will focus examining both a hydroquinone dioxygenase and an aminophenol dioxygenase enzyme in order to establish structure/activity profiles of these enzymes across a range of mutants and a set of targeted substrates. These enzymes provide an ideal platform for enzyme engineering because they are structurally characterized, easily expressed, highly active, and already fairly tolerant of different substrates. Site-directed mutagenesis, particularly targeting removing specific bulky active site amino acids will be performed to test whether this serves to broaden substrate specificity, specifically with a focus on chlorinated aromatic substrates. Steady-state kinetic characterization will be performed to define how these mutations have affected the enzyme activity, which should assist in mapping out determinants of substrate specificity. In addition, computational studies will be used to better understand the fundamental reasons why chlorinated substrates remain difficult targets for this class of enzymes. Success at generating mutant enzymes with improved activity toward substrates with different patterns of chloro-substitution has the potential to provide insight into how these enzymes bind chlorinated substrates as well as guide future efforts to engineer ring-cleaving dioxygenase enzymes. 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.