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New recipes for biocatalysis: Expanding the cytochrome P450 chemical landscape

The design and engineering of protein catalysts that carry out rare or non-natural chemistry remains a challenging contemporary goal. However, enzymes are inherently limited by their chemical composition, i.e. the reagent pool that exists in nature and the amino acids and cofactors that form their physical and catalytic core. Because of this limitation, the majority of chemical transformations developed by synthetic chemists remain, at least to our knowledge, biologically inaccessible. Biological cofactors and prosthetic groups, including heme, provide a convenient means for natural proteins to increase their range of chemical transformations. Synthetic catalysts, similar to heme, demonstrate a wealth of chemical transformations, including metallocarbene insertion reactions, through mechanisms similar to native P450 catalysis; however, these reactions do not exist in biology due to the lack of the necessary ingredients in the cell or surrounding environment. By supplementing cytochrome P450s with non-natural reagents we have been able to demonstrate that a variety of natural enzyme scaffolds are capable of carrying out reactions, including the carbene-mediated cyclopropanation of olefins, not previously observed in the natural world. Moreover, as adaptable, genetically encoded systems, the activity and product regio- and stereochemical profiles of these catalysts can be tuned through mutation. We have shown that this non-natural chemistry can be applied for fine chemical synthesis, or even adapted to modify native P450 substrates. We have gone on to show that cytochrome P450s can be evolved for the incorporation of alternative metalloporphyrin scaffolds. By combining non-natural cofactor engineering with an increase in reagent diversity, we are generating orthogonal protein systems that deliver function not available to heme containing proteins.

 

Eric Brustad is a proud native of Indianapolis, Indiana. He graduated from Purdue University (W. Lafayette, IN) in 2002 receiving B.S. degrees in Chemistry and Biology as well as a B.A. in French. As an undergraduate, he began his research career in bioorganic chemistry under the mentorship of Jean Chmielewski. In the fall of 2002, he joined The Scripps Research Institute to carry out graduate research under the direction of Peter G. Schultz. His thesis work examined applications of unnatural amino acid mutagenesis to expand protein function. Dr. Brustad moved to the California Institute of Technology in December of 2008 as a Ruth Kirschstein postdoctoral fellow where he joined the group of Frances H. Arnold. While at Caltech, Dr. Brustad applied directed protein evolution to engineer proteins for non-natural catalysis or biosensing applications. In 2012, Eric joined the Department of Chemistry and the Carolina Center for Genome Sciences at the University of North Carolina at Chapel Hill. His research program focuses on combining chemical, biological and evolutionary approaches to expand the capabilities of living cells.  His honors include the Barry M. Goldwater Scholarship (2002), a Ruth L. Kirschstein National Research Service Award (2009), a DARPA Young Faculty Award (2013), and an NSF Career Award (2015).