2 publications

2 publications

Efficient Lewis Acid Catalysis of an Abiological Reaction in a De Novo Protein Scaffold

Hilvert, D.; Jiménez-Osés, G.

Nat. Chem. 2021, 13, 231-235, 10.1038/s41557-020-00628-4

New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels–Alder reaction. The artificial metalloenzyme achieves >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M−1) that exceeds all previously characterized Diels–Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels–Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.

Metal: Zn
Ligand type: Amino acid
Host protein: De novo-designed protein
Anchoring strategy: Dative
Optimization: Genetic
Max TON: >10000
ee: 99
PDB: ---
Notes: PDB: 3V1C, 7BWW

Heteromeric Three-Stranded Coiled Coils Designed Using a Pb(ii)(Cys)3 Template Mediated Strategy

Pecoraro, V.L.

Nat. Chem. 2020, 12, 405-411, 10.1038/s41557-020-0423-6

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(ii)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(ii)A2B heterotrimers, while d sites provide pure Pb(ii)C2D or Pb(ii)CD2 scaffolds. Altering the metal from Pb(ii) to Hg(ii) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(ii)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

Metal: Pb; Zn
Ligand type: Amino acid
Host protein: De novo-designed protein
Anchoring strategy: ---
Optimization: ---
Reaction: Ester hydrolysis
Max TON: ---
ee: ---
PDB: ---
Notes: PDB: 6EGP, 6MCD