Pedro S. Coelho

Olefin Cyclopropanation via Carbene Transfer Catalyzed by Engineered Cytochrome P450 Enzymes

Putting a C in Cytochrome Cytochrome P450 enzymes oxidize hydrocarbons using a highly reactive iron oxo intermediate. Much research has focused on tuning the protein structure to broaden the range of hydrocarbons that can be functionalized. Coelho et al. (p. 307, published online 20 December; see the Perspective by Narayan and Sherman) substituted a carbene source for the oxygen to make a P450 mutant a cyclopropanation catalyst whereby a carbon fragment is transferred in place of oxygen. Though carbene activation by iron is chemically analogous to the native oxygen activation pathway, the overall reaction is completely different from any known enzymatic transformation. An oxidative enzyme has been engineered to transfer carbon to synthetic substrates in place of oxygen. [Also see Perspective by Narayan and Sherman] Transition metal–catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C–H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C–C bond–forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450BM3 that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.

A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo

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.

Enantioselective Intramolecular C-H Amination Catalyzed by Engineered Cytochrome P450 Enzymes In Vitro and In Vivo

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 P450_(BM3) have now been found to catalyze intramolecular C-H aminations in azide substrates. Mutations to two highly conserved residues significantly increased this activity.

Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity.

Synthetic Biology Approaches for Organic Synthesis

Advances in DNA technologies, metagenomics, and bioinformatics have enabled the use of biological systems (i.e., enzymes, metabolic pathways, and cells) for chemical synthesis and production of chemicals from renewable resources such as plant sugars. The authors review these basic technologies, illustrate their connection to synthetic chemistry, and provide examples of enzyme and metabolic engineering for the synthesis of organic molecules with high efficiency and selectivity. Finally, the authors anticipate the potential for increased integration of engineered enzymes in metabolic pathways as well as the creation of enzymes with completely novel activities to expand the biosynthetic capabilities.