Mentor: Dr. Jon Stewart
College of Liberal Arts and Sciences
"To further my scientific education, and to help determine a career-path."
French, Costume Design
- Stereo-Selective Enzyme Catalysis
- Actor and Stage-Fighter, Hoggetowne Medieval Faire
- Tutoring in French, English, Chemistry, History, Math
Hobbies and Interests
- Writing (mostly novels, some short fiction and poetry)
- Dance (Classical Ballet, Modern, Jazz, African, Character, etc.)
- Costume Construction (especially Corsetry)
Third-Generation Mutagenesis Studies of an Alkene Reductase
"The long-term goal of this project is to develop alkene reductases as stereoselective catalysts for eventual industrial applications, particularly the cost-effective production of pharmaceutical compounds. Alkene reductases have not been widely used in organic synthesis due to limited stereo-chemical diversity in existing enzyme collections, poor enzyme stability under process conditions, and low volumetric productivities. I will apply directed evolution strategies to Pichia stipitis OYE 2.6 in order to alter its properties, thereby solving the first two of the abovementioned problems. In previous studies, the Stewart group has applied Iterative Saturation Mutagenesis (ISM) to P. stipitis OYE 2.6, focusing on residues close to the substrate binding site (Figure 1A). The hypothesis is that these are the amino acids most likely to determine the enzyme’s substrate-acceptance and stereoselectivity. Alkenes 1 – 3 were chosen as model substrates with the goal of identifying OYE 2.6 variants with reversed stereoselectivity relative to the wild-type protein (Figure 1B). In the first round, each of the 13 targeted sites was randomized, and the resulting variants were screened against each of the three substrates. Variants that showed altered stereoselectivity against any of the three were then selected as “anchor” positions for subsequent amino acid randomizations targeted at the other positions that might also alter stereoselectivity. This process was continued for a third round with two “anchor” positions. A central tenet of directed evolution studies is that a linear pathway must connect the starting protein with the desired variants. In other words, if there is an evolutionary dead end between the starting and ending points, there is no way to cross between them. We found exactly this situation in attempts to identify an OYE 2.6 variant with reversed stereoselectivity toward alkene 3: the only way to find the best mutant (Y78W, I113C) was to pass through a single mutant (Y78W) that had no activity toward 3. The Y78W single mutant did accept substrate 2, however – the reason for which it had been retained for subsequent rounds of ISM. We suspect that there may be a similar situation for alkene 1, the one substrate for which we have yet to identify OYE 2.6 variants with altered stereoselectivity. I will therefore carry out third-generation mutagenesis studies aimed at finding variants with reversed stereoselectivity toward 2. A second key question in protein engineering revolves around the generality of the solutions: if we reverse stereoselectivity toward one substrate, does this protein variant show the same properties toward related substrates? This is a key point for synthetic utility since general solutions are much more useful than those restricted to a single compound. We will probe this, and the importance of a free hydroxyl in alkenes 1 – 3, by examining our ISM libraries for reductions of the methyl ether analogs 4 – 6 (Figure 1B). Comparing the results of these screening studies with those carried out on the free alcohols 1 – 3 will go a long way toward answering these questions."