Directed evolution of non-natural cytochrome P450 enzymes: Developing potent biocatalysts and tracing the determinants of enzyme functionality
Final Report Abstract
Recent years have seen fascinating progress in the repurposing and engineering of existing enzymes for non-natural chemical reactions. This is important, as many industrially useful chemical transformations are not catalyzed by any known enzyme, but rely on the use of often energy- and waste-intensive organic synthesis procedures. Enzymes, on the other hand, operate in aqueous solution at ambient temperatures and pressures and often provide excellent selectivities for their target reaction, potentially opening the door to efficient and “green“ biosynthetic chemical production processes. A major step towards this goal was taken by the Arnold lab and others by demonstrating the repurposing of proteins containing iron-heme cofactors for non-natural carbene and nitrene transfer reactions. These reactions rely on activated reagents and result in the formation of organic compounds with valuable chemical motifs. Among the most pliable enzymes for nonnatural reactions are hemoproteins from the cytochrome P450 family: often, only few active site mutations were sufficient to turn these enzymes into highly selective catalysts for a variety of carbene and nitrene transfer reactions. Despite these successes, these novel non-natural enzymes still lag behind their natural counterparts. The enzymatic lifetime is often limited as the enzymes are rapidly inactivated by the non-natural reagents, catalytic turnover frequency is low and the substrate scope is restricted, drastically limiting their current synthetic utility. Addressing these limitations would thus serve a dual purpose: first, improving these catalysts to the levels typically seen in natural enzymes would increase their utility for industrial synthetic applications. More importantly, insight into the factors limiting their non-natural catalytic activity and how to overcome these limitations by protein engineering would deepen our understanding of the determinants of enzymatic activity, and facilitate future engineering of proteins for non-natural reactions. During my DFG-funded time in the Arnold lab, I worked towards these goals by applying directed evolution approaches to P450 enzyme variants catalyzing non-natural carbene and nitrene transfer reactions. I first developed screening systems to efficiently identify variants with improved activity for both nitrene and carbene transfer reactions, and then utilized both random mutagenesis and site-saturation mutagenesis of rationally selected residues to engineer P450 enzymes for higher catalytic activity. In a second step, I characterized the obtained, improved enzymes biochemically and in terms of their synthetic utility with regard to substrate scope, product selectivities and yields in preparative-scale reactions. While two of three projects I worked on are currently still ongoing, the data obtained to date already provide relevant insight into the evolvability of cytochrome P450s for non-natural carbene and nitrene reactions, and the factors governing these non-natural catalytic activities.
Publications
- Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr Opin Biotechnol. 2017 Oct;47:102-111
Brandenberg OF, Fasan R, Arnold FH
(See online at https://doi.org/10.1016/j.copbio.2017.06.005)
