UC Berkeley

The following dissertation presents the development of abiotic catalysis with natural metalloproteins and artificial metalloenzymes (ArMs) by application of directed evolution and the integration of different strategies to develop ArMs.Chapter 1 provides an overview of the development of abiotic catalysis in metalloenzymes. This section presents the concept of enzymatic promiscuity, by which abiotic reactions – reactions unnatural to the protein or to biosynthetic pathways – were discovered in natural proteins and subsequently enhanced through mutagenesis. It also explores directed evolution and the strategies employed to create enzyme libraries. In addition, the chapter presents assembly strategies for incorporating metal cofactors into protein scaffolds to create artificial metalloenzymes and the abiotic reactions catalyzed by these modified enzymes. Chapter 2 focuses on the application of an abiotic zinc hydride active site in carbonic anhydrase that catalyzes the reduction of dialkyl ketones. Overcoming the inherent limitations of wild-type carbonic anhydrase, this chapter demonstrates how directed evolution and rational design were employed to generate reactive variants. Computational studies that explain the origins of enantioselectivity and substrate scope is also discussed. Chapter 3 describes the preparation of artificial metalloenzymes by covalent anchoring and dative anchoring. The first section of the chapter describes an artificial myoglobin created by covalent anchoring of a heme cofactor to the myoglobin backbone. The second section describes an optimized protocol for preparing copper-substituted carbonic anhydrase, which is characterized using various analytical approaches. The Cu-hCAII variant is shown to exhibit carbene transfer reactions that are not observed with the original carbonic anhydrase.