P450_BM3‐Axial Mutations: A Gateway to Non‐Natural Reactivity

Abstract

Enzymes capable of catalyzing non‐natural reactions have the potential to alter the way relevant molecules are prepared on‐scale. Efforts to this end have largely focused on combining non‐natural cofactors with proteins lacking catalytic function to obtain non‐natural reactivity. An alternative approach is to utilize a native cofactor to catalyze non‐natural reactions. Recently, our group demonstrated that heme‐containing cytochrome P450s are able to catalyze the highly selective cyclopropanation of alkenes. Superior activity was observed upon changing the axial cysteine to serine (“P411”). Mutation at the conserved axial ligand has enabled P450s to catalyze other non‐natural reactions such as NH insertion and CH amination.

An artificial metalloenzyme with the kinetics of native enzymes

“…To combine the favorable qualities of enzymes with the diverse reactivity of synthetic transition-metal catalysts, abiological transition-metal centers or cofactors have been incorporated into native proteins. The resulting artificial metalloenzymes catalyze classes of reactions for which there is no known enzyme (abiological transformations) (3,4).…”

Genetic Optimization of Metalloenzymes: Enhancing Enzymes for Non‐Natural Reactions

“…[14] Arnold was interested in exploring the ability of cytochrome P450s to catalyze carbene transfer reactions. [15] In as eminal report, P450 BM3 was shown to be an effective catalyst for the cyclopropanation of styrenes when using ethyl diazoacetate (EDA) as ac arbene source under anaerobic conditions,t hereby providing product with low conversion and selectivity (TTN 5; TTN = total turnover number). [16] A single mutation of the conserved distal threonine (T268), hypothesized to facilitate proton transfer in the natural reactivity,toalanine (P450 BM3 -T268A) enabled trans-selective cyclopropanation in high yield and enantioselectivity (Figure 1, top).…”

Latest Developments in Metalloenzyme Design and Repurposing

“…The groups of Arnold and Fasan have now demonstrated that formal Fe IV –carbenoid and Fe IV –nitrenoid compounds, which are isoelectronic to Fe IV –oxo, are generated in various heme proteins and insert into C–H, N–H, and S–H bonds. These C–C, C–N, and C–S bond‐forming reactions catalyzed by designed metalloenzymes are valuable, because they proceed in water, at room temperature, and are very stereoselective; this is difficult to achieve with chemical catalysts 18–20. The following carbene and nitrene transfer reactions have been described to be catalyzed by the iron porphyrin proteins: (1) carbene transfer: cyclopropanation,21–26 amination,27,28 sulfidation;29 (2) nitrene transfer: sulfamination,30–33 sulfimination,34 oxazolidinone formation 35…”