UC Berkeley

In the last decade, biocatalysis has experienced a surge in both industrial and academic researchefforts, development, and acknowledgement in the form of the Nobel Prize for directed evolution. This attention and popularity is due to the exquisite activity and selectivity of enzymes in addition to their promiscuity, which can be leveraged by the synthetic chemistry through directed evolution. Despite the advancements in directed evolution for the unnatural activity of enzymes, iterative mutagenesis can only augment a native enzyme scaffold to a point. This limitation has been addressed in part by combination of enzymes with artificial transition metal catalysts to produce artificial metalloenzymes (ArMs). The development of ArMs has also experienced a surge in recent years due to advancements in high throughput experimentation. A key limitation to ArMs, however, is that most synthetic metal cofactors are not cell-permeable. The lack of cell permeability limits experimentation with ArMs to manipulations in vitro, which severely hampers efforts to implement ArMs in directed evolution campaigns as well as employing them as part of biosynthetic pathways. In addition, few publications exist that seek to fully investigate the influence that a protein scaffold has on a metal center during the mechanism of a reaction catalyzed by ArMs. Both the lack of compatibility in vivo and mechanistic understanding of ArMs has limited the rate at which novel generations of ArM variants can be developed and studied. The work in this thesis addresses both the in vivo compatibility and mechanistic understanding of an ArM formed by the replacement of the iron-porphyrin in cytochromes P450 with an iridium porphyrin, namely the enzyme Ir(Me)-CYP119 developed by our laboratory. First, a system to assemble CYP119 within E. coli cells was developed. Next, the mechanism of cyclopropanation catalyzed by Ir(Me)-CYP119 was studied to reveal pertinent protein-cofactor dynamics and their influence on the mechanism of carbene transfer. In the final chapters, the in vivo platform was leveraged to evolve Ir(Me)-CYP119 to catalyze reactions with selectivities difficult or even impossible to achieve with small molecule catalysts. These studies have laid the foundations for and have created opportunities for nearly limitless application of hemoprotein-based ArMs to novel reactions and biosynthetic pathways in different organisms.