Novel activities and sequence-function relationships of a synthetic P450 family
Final Report Abstract
During my 2-year felllowship in Prof. Arnold’s group I worked on three projects: In my first project we developed an enzyme-based method for the selective fluorination of sites remote from functionality in a range of biologically active systems. Our technique applies both in vitro oxidation of unactivated, unfunctionalized sites in drug candidates by mutant cytochrome P450 enzymes and subsequent fluorination of the resulting hydroxyl functionality that arises from chemo-enzymatic transformation. Since methodologies that allow efficient regioselective and stereoselective late-stage fluorination of complex molecular scaffolds at sites that are remote from any functionalization have not been developed to any great extent our paper was well received in the scientific community. It was published in Nature Chemical Biology, highlighted in the News&Views section, and currently shows a factor of 4.8 (must read) in the Faculty of 1000. In my second project, we used directed evolution to extend the drug metabolite repertoire of P450BM3 variants to weakly acidic compounds which are mainly substrates of human CYP2C9 in vivo. The most active variants X3H1 and W7D8 carry up to 23 mutations compared to wild-type P450BM3 and allow conversion of the NSAIDs naproxen and ibuprofen with TTNs of 1000 and 600, respectively. We demonstrated their use in production of the respective metabolites in preparative scale (10-100 mg) in reasonable yields (up to 38%). Crystallization allowed us to elucidate the structural changes associated with the directed evolution. The mutations caused deviations in the backbone, particularly in the loop connecting helices C and D. Unlike CYP2C9, in which R108 interacts directly with the carboxylic acid group of fluorbiprofen via a salt bridge, direct coordination appears to be unlikely for 22A3 in the absence of conformational change. The introduction of R75 in the active site seems to destabilize the B’-helix which forms a large portion of the active site. However, the final mutation M185K which was found during directed evolution seems to be capable of interacting directly with naproxen according to our transition state model. Our study demonstrates that P450BM3 variants bear potential as general tools for production of drug metabolites and a viable alternative to extended screens for novel P450s from different organisms. In my third project, we addressed the bottleneck of the cellulosic biofuel production process which is the conversion of solid cellulose to fermentable sugars. We used structure-guided recombination (SCHEMA) to create novel bacterial 48 cellulases. Our library contained 6,561 chimeric proteins composed from three different parental proteins. This library comprised >3000 active bacterial family 48 cellulase chimeras, including 300 thermostable enzymes. Regression of T50 values against chimera fragment composition revealed a strong linear relationship between predicted and observed T50s for 30 tested chimeras. Our model predicts that chimeras more stable than the most stable parent protein exist in our library. We will validate our model by constructing and testing the sequences that are predicted to be most thermostable. This should allow us to create the most thermostable family 48 cellulase known to date.
Publications
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Nat Chem Biol 2009, 5, 26
A. Rentmeister, F. H. Arnold, R. Fasan
