| Summary: | Thesis (Ph. D.)--University of Rochester. Department of Chemistry, 2020. Metalloenzymes catalyze a broad range of important chemical transformations in nature and they can constitute valuable catalysts for organic synthesis. However, the reaction scope of naturally occurring metalloenzymes remains limited compared to chemical methods. Our group has recently established that myoglobin, a heme-containing protein, is a promising biocatalyst for the formation of carbon-carbon and carbon-heteroatom bonds via carbene transfer reactions, a class of synthetically valuable transformations not occurring in nature. Building upon this work, a first goal of this research was to expand and modulate the reactivity of sperm whale myoglobin-based ‘carbene transferases’ via modification of its cofactor environment. Specifically, we investigated the impact of substituting the conserved heme-coordinating histidine residue in myoglobin with both proteinogenic (Cys, Ser, Tyr, Asp) and non-proteinogenic Lewis basic amino acids (3-(3’-pyridyl)-alanine, p-aminophenylalanine, and β-(3-thienyl)-alanine), on the reactivity of this metalloprotein toward these abiotic transformations. Substitution of the proximal histidine with an aspartate residue led to a myoglobin-based catalyst capable of promoting stereoselective olefin cyclopropanation under nonreducing conditions. Next, a series of artificial myoglobin-based metalloenzymes incorporating porphyrin cofactors that contain non-native metals such as manganese, iron, cobalt, ruthenium, rhodium and iridium were investigated for cyclopropanation and Y-H (Y = N, S) carbene insertion reactions. Engineered variants containing a ruthenium cofactor were found to be excellent S-H insertion catalysts, while variants harboring Co-, Mn-, and Ir-containing cofactors were capable of C-H insertion reactions not supported by the parent protein. Finally, we demonstrated that cofactor variation in combination with mutations of the proximal ligand anchoring the metalloporphyrin in the active site pocket can drastically influence catalyst chemoselectivity. Specifically, we developed a serine-ligated cobalt-porphyrin variant that favors the more challenging olefin cyclopropanation reaction in the presence of competing and more reactive functional groups (amines and silanes). This reactivity diverges from that of the native myoglobin as well as other hemoproteins and conventional synthetic carbene transfer catalysts, which favor the more facile Y-H (Y = N, Si) insertion reaction over cyclopropanation. The second part of this PhD research was focused on stabilizing the myoglobin scaffold to retain carbene transferase activity and stereoselectivity at elevated temperatures and in the presence of chemical denaturants. To this end, we developed a new strategy for enzyme thermostabilization that relies on the installation of genetically encoded, nonreducible covalent staples in the protein using computational design. The thioether bond-forming reaction between cysteine and the genetically encodable O-2-bromoethyl-tyrosine was selected as the protein cross-linking strategy. This methodology enabled the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chemical denaturation, without affecting their catalytic efficiency and stereoselectivity properties. Building upon this study, we explored alternative unnatural amino acids for protein stapling and discovered that thioether stapling with p-(2-chloro-acetamido)-phenylalanine was effective toward yielding myoglobin-based catalysts with further improved thermostability (ΔTm: +29 °C; ΔT50: +22 °C), demonstrating the importance of subtle structural differences in the residue involved in the formation of the thioether crosslinks for protein stabilization. We also found that other noncanonical amino acids, such as p-acrylamido-phenylalanine and p-vinylsulfonamido-phenylalanine, are viable tools for protein crosslinking, illustrating the versatility of this strategy. Altogether, these studies introduced new protein engineering strategies for stabilizing protein biocatalysts as well as expanding the biocatalytic toolbox toward synthetically useful yet challenging abiological transformations.
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