Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme

Hayashi, Takahiro; Tinzl, Matthias; Mori, Takeo; Krengel, Ute; Proppe, Jonny; Soetbeer, Janne; Klose, Daniel; Jeschke, Gunnar; Reiher, Markus; Hilvert, Donald

doi:10.1038/s41929-018-0105-6

Cited by 113 publications

(146 citation statements)

References 59 publications

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“…were able to further increase the electrophilicity of the iron and thus improve the activity and selectivity of myoglobin in cyclopropanation reactions. Most remarkably, this myoglobin retained activity in absence of any reducing agent and tolerated oxygen (Table , Entry 3) …”

Section: Enzymatic Carbene‐transfer: Inspired By Organic Synthesismentioning

confidence: 97%

“… Active site residue of myoglobin variant (H64 V/V68 A) with the heme‐iron‐carbenoid complex (green). PDB: 6G5B …”

Section: Enzymatic Carbene‐transfer: Inspired By Organic Synthesismentioning

confidence: 99%

“…A milestone in this research area was uncovered in 2013 by Arnold and co‐workers, who could showcase that non‐natural cyclopropanation reactions could be achieved using an engineered enzyme . More “chemo‐inspired” reactions were uncovered over the past years which also allows the discovery of new reactions using enzymatic carbene‐transfer reactions …”

Section: Introductionmentioning

confidence: 99%

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Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes

Weissenborn

Koenigs

2020

ChemCatChem

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Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.

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Section: Enzymatic Carbene‐transfer: Inspired By Organic Synthesismentioning

confidence: 97%

“… Active site residue of myoglobin variant (H64 V/V68 A) with the heme‐iron‐carbenoid complex (green). PDB: 6G5B …”

Section: Enzymatic Carbene‐transfer: Inspired By Organic Synthesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes

Weissenborn

Koenigs

2020

ChemCatChem

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“…Given these data, we assign the new spectroscopically distinct species we describe above to the C45 and Rma-TDE metallocarbenoid intermediates. It should be noted that at this time we cannot definitively assign these spectra as either the non-bridging metallocarbenoid species observed by Lewis et al 45 or the porphyrin-bridging species observed by Hayashi et al 48 in the crystal structures of engineered cytochrome c or N-methylhistidine-ligated myoglobin variant (Mb(H64V,V68A)) respectively. However, given the identical nature of the C45 and Rma-TDE spectra, it would seem likely that the spectra obtained represent the non-bridging ligation observed in the Rma-TDE Me-EDA crystal structure 45 .…”

Section: Resultsmentioning

confidence: 63%

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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“…126 These efforts are complemented by the mechanistic work of other groups, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 further our understanding of how iron porphyrins and heme proteins catalyze carbene transfer to olefins, N-H, and C-H bonds. [127][128][129][130][131][132][133] Genetically programmable chiral organoborane synthesis was also realized by the laboratory evolution of Rma cyt c. 123 This platform successfully yields structurally distinct organoboranes through divergent directed evolution (Scheme 4B), providing bacterial borylation catalysts that are suitable for gram-scale biosynthesis, and offering up to 15,300 turnovers and excellent selectivity (99:1 e.r., 100% chemoselectivity). Moreover, these enzymes are readily tunable to accommodate the synthesis of lactonebased organoboranes 134 and a broad range of chiral α-trifluoromethylated organoboranes (Scheme 4C).…”

Section: New Chemical Bondsmentioning

confidence: 99%

A Continuing Career in Biocatalysis: Frances H. Arnold

2019

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On the occasion of Professor Frances H. Arnold's recent acceptance of the 2018 Nobel Prize in Chemistry, we honor her numerous contributions to the fields of directed evolution and biocatalysis. Arnold pioneered the development of directed evolution methods for engineering enzymes as biocatalysts. Her highly interdisciplinary research has provided a ground not only for understanding the mechanisms of enzyme evolution but also for developing commercially viable enzyme biocatalysts and biocatalytic processes. In this Account, we highlight some of her notable contributions in the past three decades in the development of foundational directed evolution methods and their applications in the design and engineering of enzymes with desired functions for biocatalysis. Her work has created a paradigm shift in the broad catalysis field.

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Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme

Cited by 113 publications

References 59 publications

Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes

Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

A Continuing Career in Biocatalysis: Frances H. Arnold

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