9G52

Crystal structure of LmrR with V15 replaced by unnatural amino acid 4-mercaptophenylalanine, Au(I) bound

This is a non-PDB format compatible entry.

Summary for 9G52

• Entry DOI: 10.2210/pdb9g52/pdb
• Descriptor: Transcriptional regulator, PadR-like family, GOLD ION (3 entities in total)
• Functional Keywords: artificial metalloenzyme, unnatural amino acid, 4-mercaptophenylalanine, gold-bound, metal binding protein
• Biological source: Lactococcus cremoris subsp. cremoris MG1363
• Total number of polymer chains: 2
• Total formula weight: 29886.76

Authors

Thunnissen, A.M.W.H.,Aalbers, F.S.,Veen, M.J.,Rozeboom, H.J.,Roelfes, G. (deposition date: 2024-07-16, release date: 2024-12-25, Last modification date: 2025-03-26)

Primary citation

Veen, M.J.,Aalbers, F.S.,Rozeboom, H.J.,Thunnissen, A.W.H.,Sauer, D.F.,Roelfes, G.
Artificial Gold Enzymes Using a Genetically Encoded Thiophenol-Based Noble-Metal-Binding Ligand.
Angew.Chem.Int.Ed.Engl., 64:e202421912-e202421912, 2025

PubMed 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.

PubMed: 39629678
DOI: 10.1002/anie.202421912

Experimental method

X-RAY DIFFRACTION (2.5 Å)