A Dual‐Metal‐Catalyzed Sequential Cascade Reaction in an Engineered Protein Cage**

Abstract

Herein, we describe the creation of an artificial protein cage housing a dual-metal-tagged guest protein that catalyzes a linear, two-step sequential cascade reaction. The guest protein consists of a fusion protein of HaloTag and monomeric rhizavidin. Inside the protein capsid, we established a ruthenium-catalyzed allylcarbamate deprotection reaction followed by a goldcatalyzed ring-closing hydroamination reaction that led to indoles and phenanthridines with an overall yield of up to 66 % in aqueous solutions. Furthermore, we show that the encapsulation stabilizes the metal catalysts against deactivation by air, proteins and cell lysate.

Pore engineering as a general strategy to improve protein-based enzyme nanoreactor performance

“…This includes both in vivo applications related to metabolic engineering 2 and bioremediation 3 , in vitro applications as chemoenzymatic production systems 4 , and applications as delivery devices for biomedically relevant enzymes 5 . Encapsulating enzymes inside nanoreactors can result in a number of distinct advantages, including the controllable co-localization of enzymes 6 , the selective enrichment of substrates 7 , intermediate sequestration and channeling 8 , prevention of unwanted side reactions and toxicity 9 , increased enzyme stability and protease resistance [10][11][12][13] , and the ability to sequester and store molecules of interest in a soluble and non-toxic form 14 . Depending on the system, nanoreactors are often assembled in vitro using relatively harsh disassembly, mixing, and reassembly protocols [15][16][17][18] .…”

“…BMCs have been engineered for ethanol 36 and hydrogen 37 production, in both cases enzymatic activity decreased upon encapsulation. Due to their robust plug-and-play cargo loading mechanism, encapsulins have also been engineered for nanoreactor applications including for phenanthridine 11 and 5-methyltetrahydrofolate 38 production, luciferase encapsulation 12 , and alcohol and oligosaccharides oxidation 13 . However, consistently, as the size of substrates increased, the catalytic activity of encapsulated enzymes significantly decreased.…”