LongLarf

The design of artificial enzymes represents a transformative advancement in biocatalysis, enabling the creation of enzyme for nonnatural reactions. Herein, recent progress in the design of enzymes is highlighted featuring unnatural catalytic residues introduced via site-specific chemical modification. This concept emphasizes the methodologies employed, the challenges, and future directions for expanding potential applications of artificial enzyme design in biocatalysis.

The design of artificial enzymes represents a transformative advancement in biocatalysis, enabling the creation of bespoke biocatalysts for nonnatural reactions. A key innovation in this field is the introduction of unnatural catalytic residues through site-specific chemical modification, which significantly expands the chemical repertoire of natural enzymes. This approach combines precision engineering with cutting-edge methodologies, including chemical ligation, noncanonical amino acid incorporation and directed evolution. These strategies facilitate the development of enzymes with novel catalytic activities, modify substrate specificities, and enhance stability under nonphysiological conditions. This concept examines the methodologies, challenges, and future directions in the design of enzymes with unnatural catalytic residues via site-specific chemical modification, with a focus on their functional impact and transformative potential in synthetic chemistry and biocatalysis.

The generic incorporation of a thioxanthone-containing amino acid into a protein scaffold is described. The resulting artificial photoenzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields with excellent enantioselectivity.

Abstract

Genetically encodable photosensitizers allow the design of artificial photoenzymes to expand the scope of abiological reactions. Herein, we report the genetic incorporation of a thioxanthone-containing amino acid into a protein scaffold via an engineered pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase pair. The designer enzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields (up to>99 % yield) with excellent enantioselectivity (up to 98 : 2 e.r.). This work provides a robust and facile method for photoenzyme design and lays the foundation for the development of further photoenzymatic reactions.

Nature Catalysis, Published online: 11 December 2024; doi:10.1038/s41929-024-01259-5

A new class of artificial metalloenzymes containing genetically encoded noble-metal-binding sites featuring a non-canonical thiophenol-based amino acid, which serves as an excellent soft ligand for binding various noble metals, was developed. The corresponding gold(I) enzyme was characterised, confirming gold binding to the thiophenol, and successfully applied in catalytic hydroamination reactions.

Abstract

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 through 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 turnover numbers over 50. 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 observed, 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 for 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.

Nature, Published online: 20 November 2024; doi:10.1038/s41586-024-08327-7

Nature, Published online: 13 November 2024; doi:10.1038/s41586-024-08186-2