An Artificial Heme Enzyme for Cyclopropanation Reactions

Villarino, Lara; Splan, Kathryn E.; Madan, Bharat; Alonso‐Cotchico, Lur; Souza, Cora Gutiérrez de; Lledós, Agustı́; Maréchal, Jean‐Didier; Thunnissen, A.M.W.H.; Roelfes, Gérard

doi:10.1002/anie.201802946

Cited by 118 publications

(102 citation statements)

References 42 publications

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“…In particular, C45/EDA catalyzed cyclopropanation of p-trifluoromethylstyrene and p-methoxystyrene occur with exceptional yield and enantioselectivity for the (R,R) product (99.7% yield & 91.4% ee for F3C-substituted; 90.4% yield & 92.8% ee for MeO-substituted). We postulate that the observed stereoselectivity exhibited by C45 results from the intrinsic asymmetry of the protein scaffold, likely through specific positioning of the reactive carbene adduct as proposed by Villarino et al 24 , and more recently observed in the crystal structure of the methyl-EDA adduct of the engineered cytochrome c 45 . It also offers a means by which we can improve subsequent C45-derived maquettes.…”

Section: Resultssupporting

confidence: 56%

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

Stenner

Steventon

Seddon

et al. 2018

Preprint

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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 the first report of carbene transferase activity in a de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.Despite the significant advances in protein design, there still remain few examples of de novo enzymes that both approach the catalytic efficiencies of their natural counterparts and are of potential use in an industrial or biological context [1][2][3][4][5][6][7] . This reflects the inherent complexities experienced in the biomolecular design process, where approaches are principally focussed on either atomistically-precise redesign of natural proteins to stabilise reaction transition states 1-3 or imprinting the intrinsic chemical reactivity of cofactors or metal ions on simple, generic protein scaffolds 4-7 ; both can be significantly enhanced by implementing powerful, yet randomised directed evolution strategies to hone and optimise incipient function 8,9 . While the latter approach often results in de novo proteins that lack a singular structure 4,10,11 , the incorporation of functionally versatile cofactors, such as heme, is proven to facilitate the design and construction of de novo proteins and enzymes that recapitulate the function of natural heme-containing proteins in stable, simple and highly mutable tetrahelical chassis (termed maquettes) [12][13][14] . Since the maquettes are designed from first principles, they lack any natural evolutionary history and the associated functional interdependency that is associated with natural protein scaffolds can be largely circumvented 12 .We have recently reported the design and construction of a hyperthermostable maquette, C45, that is wholly assembled in vivo, hijacking the natural E. coli cytochrome c maturation system to covalently append heme onto the protein backbone (Fig. 1a) 4 . The covalently-linked heme C of C45 is axially ligated by a histidine side chain at the proximal site and it is likely that a water molecule occupies the distal site, analogous to the ligation state of natural heme-containing peroxidases 15 and metmyoglobin 16 . Not only does C45 retain the reversible oxygen binding capability of its ancestral maquettes 4,12,13 , but it functions as a promiscuous and catalytically proficient peroxidase, catalyzing the oxidation of small molecules, redox proteins and the oxidative dehalogenation of halogenated phenols with kinetic parameters that match and even surpass those of natural peroxidases 4 .

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Section: Resultssupporting

confidence: 56%

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

Stenner

Steventon

Seddon

et al. 2018

Preprint

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“…This result was achieved by a conservative introduction of only two changes into the sequence of a catalytic amyloid-forming peptide incapable of binding hemin. The efficiencies of these catalysts are comparable to some engineered proteins [11] and demonstrate the ease with which hemin can be employed to promote cyclopropanation in general; however, the observed enantioselectivities are modest, consistent with the lack of a well-defined enclosed catalytic site found in globular proteins. Nonetheless, it is quite striking that self-assembly of short peptides into essentially flat surfaces results in any enantioselectivity at all and this observation suggests the subtle effects of the assembly on the transition state of the reaction.…”

Section: Angewandte Chemiesupporting

confidence: 53%

Semi‐Rationally Designed Short Peptides Self‐Assemble and Bind Hemin to Promote Cyclopropanation

Zozulia

Korendovych

2020

Angewandte Chemie

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The self-assembly of short peptides gives rise to versatile nanoassemblies capable of promoting efficient catalysis. We have semi-rationally designed a series of seven-residue peptides that form hemin-binding catalytic amyloids to facilitate enantioselective cyclopropanation with efficiencies that rival those of engineered hemin proteins. These results demonstrate that: 1) Catalytic amyloids can bind complex metallocofactors to promote practically important multisubstrate transformations. 2) Even essentially flat surfaces of amyloid assemblies can impart a substantial degree of enantioselectivity without the need for extensive optimization.3) The ease of peptide preparation allows for straightforward incorporation of unnatural amino acids and the preparation of peptides made from d-amino acids with complete reversal of enantioselectivity.

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“…To identify beneficial mutations,e ight residues were targeted (Supporting Information, Figure S2), which are either in close proximity to pAF15 or have previously been identified to improve catalytic parameters of unrelated, LmrR-based designer enzymes. [32][33][34] Libraries targeting each position were constructed using degenerate primers (NNK codons,w hich allow for all 20 canonical amino acids) and approximately 400 library members were evaluated using the previously established screen.…”

Section: Zuschriftenmentioning

confidence: 99%

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

et al. 2019

Self Cite

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The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency ( k cat ) of the designer enzyme by almost 100‐fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200‐fold.

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An Artificial Heme Enzyme for Cyclopropanation Reactions

Cited by 118 publications

References 42 publications

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

Semi‐Rationally Designed Short Peptides Self‐Assemble and Bind Hemin to Promote Cyclopropanation

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

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