Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a unique serine-heme ligated cytochrome “P411” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450BM3 gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state FeIII/II reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity.

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe.

Nitrogen activation: Though P450 enzymes are masters of oxygen activation and insertion into CH bonds, their ability to use nitrogen for the same purpose has so far not been explored. Engineered variants of cytochrome P450BM3 have now been found to catalyze intramolecular CH aminations in azide substrates. Mutations to two highly conserved residues significantly increased this activity.

The ability of supramolecular host-guest complexes to catalyse organic reactions collaboratively with an enzyme is an important goal in the research and discovery of synthetic enzyme mimics. Herein we present a variety of catalytic tandem reactions that employ esterases, lipases or alcohol dehydrogenases and gold(I) or ruthenium(II) complexes encapsulated in a Ga(4)L(6) tetrahedral supramolecular cluster. The host-guest complexes are tolerated well by the enzymes and, in the case of the gold(I) host-guest complex, show improved reactivity relative to the free cationic guest. We propose that supramolecular encapsulation of organometallic complexes prevents their diffusion into the bulk solution, where they can bind amino-acid residues on the proteins and potentially compromise their activity. Our observations underline the advantages of the supramolecular approach and suggest that encapsulation of reactive complexes may provide a general strategy for carrying out classic organic reactions in the presence of biocatalysts.