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. 2014 Jun 23;53(26):6810-3.
doi: 10.1002/anie.201402809. Epub 2014 May 6.

Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran

Z Jane Wang  1 , Hans Renata, Nicole E Peck, Christopher C Farwell, Pedro S Coelho, Frances H Arnold
Affiliations

Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran

Z Jane Wang et al. Angew Chem Int Ed Engl. .

Abstract

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.

Keywords: biocatalysis; cyclopropanation; cytochrome P450; enzymes; protein engineering.

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Figures

Figure 1
Figure 1
(a) Ser-ligated heme in BM3enablescyclopropanationin vivo. (b) Proposed Ala-, Met-, His-, and Tyr-ligated heme in AxX variants of BM3.
Figure 2
Figure 2
Reaction progress of in vivo cyclopropanation of styrene with EDA catalyzed by axial variants of T268A.
Figure 3
Figure 3
Sequential site saturation mutagenesis at key active site positions in T268A-AxH variant led to increased enantioselectivity for cyclopropanation of 1.
Scheme 1
Scheme 1
Proposed formal synthesis of levomilnacipran using P450-catalyzed enantioselective cyclopropanation in the key ring-forming step.
See this image and copyright information in PMC

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