A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase

Richard Stenner,Jack W Steventon,Annela Seddon,J L Ross Anderson

doi:10.1073/pnas.1915054117

Richard Stenner, Jack W Steventon + Show 2 more

Open Access

https://doi.org/10.1073/pnas.1915054117

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Abstract

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

Highlights

| | | de novo protein design enzyme design carbene transfer biocatalytic | ring expansion biocatalysis
We have showcased the exceptionally diverse functionality available in this simple, de novo-designed heme protein and demonstrated that the carbene transferase activity intrinsic to the scaffold compares very favorably to that reported for most engineered natural proteins
The formation of the metallocarbenoid intermediate at the C45 heme facilitates the high-yielding cyclopropanation of styrene and its derivatives, and extends to the insertion of carbenes into N–H bonds, the olefination of carbenes, and a description of enzymecatalyzed ring expansion of a nitrogen heterocycle. This demonstrates that abiological function is intrinsic to these de novodesigned maquettes and that the higher complexity of natural protein frameworks, such as the globins and cytochromes P450, is not necessary to support reactivity of this type

Summary

Results and Discussion

Generation of Reactive Metallocarbenoids in the De Novo-Designed C45 and an Engineered Cytochrome c (Rma-TDE). To confirm the identity of the spectroscopically isolated species as the putative metallocarbenoid intermediate, we rapidly mixed ferrous C45 with 100 μM EDA and 3 mM styrene at 5 °C in a stopped-flow spectrophotometer (Fig. 3) Under these conditions, the same putative intermediate spectrum appeared over 60 s, but decayed slowly to the starting ferrous C45 spectrum, consistent with the proposed mechanism of heme-catalyzed carbene transfer to the olefin, in which there is no net transfer of electrons from the heme [47, 48]. While the rate of metallocarbenoid intermediate and subsequent product formation appeared relatively low in our stopped-flow experiments, it is worth noting that the selected conditions were necessary for maximizing the quantity of intermediate in the stopped-flow apparatus and that subsequent activity assays were carried out at higher substrate concentrations (both EDA and styrene), higher temperature, and lower ethanol concentrations This would undoubtedly lead to higher reaction rates than those presented in the stopped-flow data here.

Carbene adduct then product formation

Conclusions

Experimental Details