9H9W

Crystal structure of metal-free LmrR_V15Bpy in an open state

Summary for 9H9W

• Entry DOI: 10.2210/pdb9h9w/pdb
• Descriptor: Transcriptional regulator, PadR-like family, 2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL (3 entities in total)
• Functional Keywords: artificial metalloenzyme, unnatural amino acid, bipyridine, copper-binding, lmrr, metal binding protein
• Biological source: Lactococcus cremoris subsp. cremoris MG1363
• Total number of polymer chains: 2
• Total formula weight: 30546.43

Authors

Thunnissen, A.M.W.H.,Jiang, R.,Casilli, F.,Aalbers, F.,Roelfes, G. (deposition date: 2024-11-01, release date: 2025-03-05)

Primary citation

Jiang, R.,Casilli, F.,Thunnissen, A.W.H.,Roelfes, G.
An artificial copper-Michaelase featuring a genetically encoded bipyridine ligand for asymmetric additions to nitroalkenes.
Angew.Chem.Int.Ed.Engl., :e202423182-e202423182, 2025

PubMed Abstract: Artificial metalloenzymes (ArMs) are an attractive approach to achieving "new to nature" biocatalytic transformations. In this work, a novel copper-dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper-binding ligand, i. e. (2,2-bipyridin-5-yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non-canonical metal-binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper-catalyzed asymmetric addition of 2-acetyl azaarenes to nitroalkenes, yielding various γ-nitro butyric acid derivatives, which are precursors for a range of high-value-added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99 % yield and 99 % ee). Additionally, the evolved variant could be further used in a preparative-scale synthesis, providing chiral products for diverse derivatizations. X-ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2-acetyl azaarene substrate to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent K for both substrates and a notable threefold increase in apparent k for 2-acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non-canonical metal-binding ligands for new-to-nature biocatalysis.

PubMed: 39945539
DOI: 10.1002/anie.202423182

Experimental method

X-RAY DIFFRACTION (2.26 Å)