4 publications

4 publications

Directed Evolution of Artificial Metalloenzymes: Bridging Synthetic Chemistry and Biology

Review

Arnold, F.H.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 137-170, 10.1002/9783527804085.ch5

Directed evolution is a powerful algorithm for engineering proteins to have novel and useful properties. However, we do not yet fully understand the characteristics of an evolvable system. In this chapter, we present examples where directed evolution has been used to enhance the performance of metalloenzymes, focusing first on “classical” cases such as improving enzyme stability or expanding the scope of natural reactivity. We then discuss how directed evolution has been extended to artificial systems, in which a metalloprotein catalyzes reactions using abiological reagents or in which the protein utilizes a nonnatural cofactor for catalysis. These examples demonstrate that directed evolution can also be applied to artificial systems to improve catalytic properties, such as activity and enantioselectivity, and to favor a different product than that favored by small‐molecule catalysts. Future work will help define the extent to which artificial metalloenzymes can be altered and optimized by directed evolution and the best approaches for doing so.


Notes: Book chapter

Exploiting and Engineering Hemoproteins for Abiological Carbene and Nitrene Transfer Reactions

Review

Arnold, F.H.; Fasan, R.

Curr. Opin. Biotechnol. 2017, 47, 102-111, 10.1016/j.copbio.2017.06.005

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle–Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities.


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Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Ligands

Arnold, F.H.

J. Am. Chem. Soc. 2019, 141, 19585-19588, 10.1021/jacs.9b11608

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that Pseudomonas savastanoi ethylene-forming enzyme, a non-heme iron enzyme, can catalyze olefin aziridination and nitrene C−H insertion, and that these activities can be improved by directed evolution. The nonheme iron center allows for facile modification of the primary coordination sphere by addition of metalcoordinating molecules, enabling control over enzyme activity and selectivity using small molecules.


Metal: Fe
Ligand type: Amino acid
Anchoring strategy: Native
Optimization: Genetic
Reaction: C-H amination
Max TON: 730
ee: 61
PDB: 6CBA
Notes: Additional reaction: aziridination

Selective C–H Bond Functionalization with Engineered Heme Proteins: New Tools to Generate Complexity

Review

Arnold, F.H.

Curr. Opin. Chem. Biol. 2019, 49, 67-75, 10.1016/j.cbpa.2018.10.004

C–H functionalization is an attractive strategy to construct and diversify molecules. Heme proteins, predominantly cytochromes P450, are responsible for an array of C–H oxidations in biology. Recent work has coupled concepts from synthetic chemistry, computation, and natural product biosynthesis to engineer heme protein systems to deliver products with tailored oxidation patterns. Heme protein catalysis has been shown to go well beyond these native reactions and now accesses new-to-nature C–H transformations, including C–N and C–C bond forming processes. Emerging work with these systems moves us along the ambitious path of building complexity from the ubiquitous C–H bond.


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