Expanding the Scope of Metalloprotein Families and Substrate Classes in New-to-Nature Reactions

Abstract

Heme proteins, in particular cytochromes P450, have been extensively used in biocatalytic applications due to their high degree of regio-, chemo-, and stereoselectivity in oxene-transfer reactions. In 2013, it was shown for the first time that engineered heme proteins can also catalyze analogous carbene- and nitrene-transfer reactions. Research in this field has since grown dramatically, with emphasis on developing new heme protein variants to increase the scope of biotransformations accessible through these new transfer reactions. This thesis details the expansion of these new-to-nature carbene and nitrene-transfer reactions to include new substrate classes previously unexplored with iron-porphyrin proteins, the use of non-heme metalloproteins for these transformations, and steps toward improving the robustness of the new-to-nature biocatalytic platform. Chapter 1 introduces the steps the field of biocatalysis has taken toward engineering enzymes with new catalytic functions and the process by which these activities are discovered and enhanced. Chapter 2 details the discovery and engineering of heme proteins which catalyze the stereodivergent cyclopropanation of unactivated and electron-deficient alkenes via carbene transfer, expanding the substrate classes beyond styrenyl alkenes. Chapter 3 shows the development of engineered variants of a heme protein (Rhodothermus marinus nitric oxide dioxygenase) for the diastereodivergent synthesis of cyclopropanes functionalized with a pinacolborane moiety, enabling product diversification through standard cross-coupling reactions. In Chapter 4, a collection of non-heme metalloproteins is curated, and a non-heme iron enzyme (Pseudomonas savastanoi ethylene-forming enzyme) is shown to be both amenable to directed evolution and non-native ligand substitution to enhance its nitrene-transfer activity. Chapter 5 describes the expansion of sequence space targeted for screening in the serine-ligated cytochrome P411 from Bacillus megaterium (P411BM3) biocatalytic platform to enhance the mutational robustness of these remarkable enzymes. Overall, this work provides a framework for bringing model new-to-nature reactions to their full potential in synthetic biocatalytic reactions.