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

Area of Science:

• Bioinorganic Chemistry
• Enzyme Engineering
• Catalysis

Background:

• Incorporating noble metals into artificial metalloenzymes (ArMs) is hindered by the limited availability of suitable soft coordinating ligands in natural amino acids.
• Developing ArMs with noble metal active sites requires novel strategies for ligand design and metal incorporation.

Purpose of the Study:

• To engineer a new class of ArMs with a genetically encoded noble-metal-binding site.
• To utilize a non-canonical thiophenol-based amino acid, 4-mercaptophenylalanine (pSHF), as a soft ligand for noble metals.
• To demonstrate the catalytic activity of the resulting gold(I) ArM in hydroamination reactions.

Main Methods:

• Genetically encoding 4-mercaptophenylalanine (pSHF) into the transcriptional regulator LmrR using stop codon suppression.
• Characterization of the gold(I) ArM using mass spectrometry, UV/Vis spectroscopy, and X-ray crystallography.
• Site saturation mutagenesis and on-bead screening for ArM optimization.

Main Results:

• The pSHF incorporated into LmrR serves as an effective ligand for gold(I) in low oxidation states.
• The gold(I) ArM successfully catalyzed hydroamination reactions of 2-ethynyl anilines with turnover numbers exceeding 50.
• High regioselectivity (up to 98%) was achieved in the hydroamination of an ethynylphenylurea substrate, yielding the phenyl-dihydroquinazolinone product.
• Two equivalents of gold(I) per protein dimer were required for optimal activity.
• Site-directed mutagenesis identified a single mutant with enhanced activity for specific substrates.

Conclusions:

• Thiophenol-based amino acids are viable alternative ligands for noble metals in enzyme engineering.
• This work expands the scope of noble metal catalysis within the field of artificial metalloenzymes.
• The developed ArM offers new possibilities for coordination chemistry and catalytic applications.