Directed evolution of the substrate specificity of dialkylglycine decarboxylase.

Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~2.5-fold decrease in KM and ~2-fold increase in kcat, giving a modest ~5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

Using invention to change how students tackle problems.

Invention activities challenge students to tackle problems that superficially appear unrelated to the course material but illustrate underlying fundamental concepts that are fundamental to material that will be presented. During our invention activities in a first-year biology class, students were presented with problems that are parallel to those that living cells must solve, in weekly sessions over a 13-wk term. We compared students who participated in the invention activities sessions with students who participated in sessions of structured problem solving and with students who did not participate in either activity. When faced with developing a solution to a challenging and unfamiliar biology problem, invention activity students were much quicker to engage with the problem and routinely provided multiple reasonable hypotheses. In contrast the other students were significantly slower in beginning to work on the problem and routinely produced relatively few ideas. We suggest that the invention activities develop a highly valuable skill that operates at the initial stages of problem solving.