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

Abstract

Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe(III)-to-Fe(II) reduction potential and substantially improves NAD(P)H-driven activity.

Figure 1

Contrasting P450- and P411-mediated cyclopropanation. (A) Cytochrome P450s inefficiently catalyze cyclopropanation using NAD(P)H as a reductant because the Fe^III/Fe^II redox potential for the low-spin resting state (E°′_Fe-cys = −430 mV) is lower than that of NAD(P)⁺/NAD(P)H (E°′₁ = −320 mV, right). Mutation of the heme-ligating Cys to Ser allows NAD(P)H-driven cyclopropanation while removing native monooxygenation (left). (B) Close-up of the P411_BM3-heme-CIS active site (PDB: 4H24) superimposed with an F_o - F_c simulated annealing omit map contoured at 3 σ showing electron density (green mesh) corresponding to the bound heme and C400S mutation. Heme, C400S and additional active site amino acid side chains are shown as sticks. (C) In vitro cyclopropanation vs. epoxidation of styrene catalyzed by P450_BM3-CIS and P411_BM3-CIS under anaerobic and aerobic conditions. Reaction conditions were as follows: 30 mM styrene, 10 mM EDA, 0.5 mM NADPH, 25 mM glucose, 2 U ml⁻¹ glucose dehydrogenase and 20 μM enzyme in aqueous potassium phosphate buffer and 5% MeOH cosolvent for six hours at 25 °C. Error bars represent the standard deviation of three independent measurements.