BIOPHYSICS SEMINAR<br>Watching Enzymes at Work: NMR for Structural and Functional Studies of Cytochromes P450

Our group is interested in how biomolecules such as enzymes and proteins do their jobs.  Nature has had a very long time (4 billion years!) to come up with just the right combinations of sequence and fold to accomplish all of the biochemical tasks required by a living organism.  We don't have quite that long, so we use a variety of biophysical and molecular biological methods to try and tease out the details of protein structure and function.  Our aim is to be able to apply what we learn to such things as rational drug design and protein engineering.
Cytochrome P450 monooxygenases catalyze the selective oxidation of unactivated C-H and C-C bonds by molecular oxygen, and this activity is critical for diverse biological functions including steroid hormone biosynthesis, drug and xenobiotic metabolism and clearance, and pro-drug activation.  We are the first group to apply high-resolution NMR methods to understanding structure-activity relationships in the P450 superfamily.  NMR offers a unique perspective on these enzymes, as it allows atomic-resolution detail of the enzymes as they exist in solution.  The P450 reaction cycle is complicated, requiring multiple substrate binding, electron and proton transfer steps, with interactions between redox partners and effector molecules leading to poorly understood conformational changes.  We have identified critical (and previously unrecognized) conformational changes that occur during the course of the reaction cycle, and have pinpointed particular residues that are involved in those changes using NMR and mutagenesis.  We are currently using a combination of enzyme activity assays, site-directed mutagenesis and NMR to “evolve”  new P450 enzymes based on the well-understood cytochrome P450cam (Figure 1).  Our aim is to produce new enzymes that combine the efficiency and selectivity of the wild-type enzyme with modified substrate selectivity/product specificity.  We envision a family of enzymes, each with its own particular substrate/product combination, that can be used for environmentally friendly manufacturing of fine chemicals.