Olefin cyclopropanation and carbon-hydrogen amination via carbene and nitrene transfers catalyzed by engineered cytochrome P450 enzymes

Synthetic biology promises to transform organic synthesis by enabling artificial catalysis in living cells. I start by reviewing the state of the art in this young field and recognizing that new approaches are required for designing enzymes that catalyze nonnatural reactions, in order to expand the scope of biocatalytic transformations. Carbene and nitrene transfers to C=C and C-H bonds are reactions of tremendous synthetic utility that lack biological counterparts. I show that various heme proteins, including cytochrome P450BM3, will catalyze promiscuous levels of olefin cyclopropanation when provided with the appropriate synthetic reagents (e.g., diazoesters and styrene). Only a few amino acid substitutions are required to install synthetically useful levels of stereoselective cyclopropanation activity in P450BM3. Understanding that the ferrous-heme is the active species for catalysis and that the artificial reagents are unable to induce a spin-shift-dependent increase in the redox potential of the ferric P450, I design a high-potential serine-heme ligated P450 (P411) that can efficiently catalyze cyclopropanation using NAD(P)H. Intact E. coli whole-cells expressing P411 are highly efficient asymmetric catalysts for olefin cyclopropanation. I also show that engineered P450s can catalyze intramolecular amination of benzylic C-H bonds from arylsulfonyl azides. Finally, I review other examples of where synthetic reagents have been used to drive the evolution of novel enzymatic activity in the environment and in the laboratory. I invoke preadaptation to explain these observations and propose that other man-invented reactions may also be transferrable to natural enzymes by using a mechanism-based approach for choosing the enzymes and the reagents. Overall, this work shows that existing enzymes can be readily adapted for catalysis of synthetically important reactions not previously observed in nature.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions

The creation of novel enzyme activity is a great challenge to protein engineers, but nature has done so repeatedly throughout the process of natural selection. I begin by outlining the multitude of distinct reactions catalyzed by a single enzyme class, cytochrome P450 monooxygenases. I discuss the ability of cytochrome P450 to generate reactive intermediates capable of diverse reactivity, suggesting this enzyme can also be used to generate novel reactive intermediates in the form of metal-carbenoid and nitrenoid species. I then show that cytochrome P450 from Bacillus megaterium (P450_BM3) and its isolated cofactor can catalyze metal-nitrenoid transfer in the form of intramolecular C–H bond amination. Mutations to the protein sequence can enhance the reactivity and selectivity of this transformation significantly beyond that of the free cofactor. Next, I demonstrate an intermolecular nitrene transfer reaction catalyzed by P450_BM3 in the form of sulfide imidation. Understanding that sulfur heteroatoms are strong nucleophiles, I show that increasing the sulfide nucleophilicity through substituents on the aryl sulfide ring can dramatically increase reaction productivity. To explore engineering nitrenoid transfer in P450BM3, active site mutagenesis is employed to tune the regioselectivity intramolecular C–H amination catalysts. The solution of the crystal structure of a highly selective variant demonstrates that hydrophobic residues in the active site strongly modulate reactivity and regioselectivity. Finally, I use a similar strategy to develop P450-based catalysts for intermolecular olefin aziridination, demonstrating that active site mutagenesis can greatly enhance this nitrene transfer reaction. The resulting variant can catalyze intermolecular aziridination with more than 1000 total turnovers and enantioselectivity of up to 99% ee.