RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Public Deposited

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  • March 20, 2019
Creator
  • Gober, Joshua
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach.
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Rights statement
  • In Copyright
Advisor
  • Brustad, Eric
  • Li, Bo
  • Johnson, Jeffrey
  • Pielak, Gary J.
  • Waters, Marcey
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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