Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study

Klemencic, E.; Brewster, R. C.; Ali, H. S.; Richardson, J. M.; Jarvis, A. G.

doi:10.1039/d3cy01648j

Catal. Sci. Technol.

2024

DOI: 10.1039/d3cy01648j

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Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study

E. Klemencic,

R. C. Brewster,

H. S. Ali

et al.

Abstract: Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein...

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Cited by 11 publications

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A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

Day,

Zaytsev,

Brewster

et al. 2024

Angewandte Chemie

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A rationally designed dual purpose non‐canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold via genetic code expansion. Trz contains a 5‐pyridyl‐1,2,4‐triazine system, which allows for inverse electron‐demand Diels–Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site‐specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)‐bound protein blue‐shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual purpose non‐canonical amino acid, which has great potential for myriad bioapplications where metal‐based functionality is required, for example in imaging, catalysis, or photo‐dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual modality imaging or therapeutic payloads.

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A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

Day,

Zaytsev,

Brewster

et al. 2024

Angewandte Chemie

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Artificial Gold Enzymes Using a Genetically Encoded Thiophenol‐Based Noble‐Metal‐Binding Ligand

Veen,

Aalbers,

Rozeboom

et al. 2024

Angewandte Chemie

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Incorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble‐metal‐binding site based on a non‐canonical thiophenol‐based amino acid, 4‐mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR via stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states. The corresponding gold(I) enzyme was characterised by mass spectrometry, UV‐vis spectroscopy and X‐ray crystallography and successfully catalysed hydroamination reactions of 2‐ethynyl anilines with over 50 turnover numbers. Interestingly, two equivalents of gold(I) per protein dimer proved to be required for activity. Up to 98% regioselectivity in the hydroamination of an ethynylphenylurea substrate was obtained, yielding the corresponding phenyl‐dihydroquinazolinone product, consistent with a π‐activation mechanism by single gold centres. The ArM was optimized by site saturation mutagenesis, using an on‐bead screening protocol. This resulted in a single mutant that showed higher activity on one class of substrates. This work brings the power of noble‐metal catalysis into the realm of enzyme engineering and establishes thiophenols as alternative ligands for noble metals, providing new opportunities in coordination chemistry and catalysis.

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A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

Day,

Zaytsev,

Brewster

et al. 2024

Angew Chem Int Ed

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Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study

Abstract: Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein...

Cited by 11 publications

References 42 publications

A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

Artificial Gold Enzymes Using a Genetically Encoded Thiophenol‐Based Noble‐Metal‐Binding Ligand

A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

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