Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design
J. Am. Chem. Soc. 2015, 137, 10414-10419, 10.1021/jacs.5b06622
Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η5-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5–), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46–64-fold improved affinity for the iridium pianostool complex [(η5-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.
Metal: IrLigand type: Cp*; Pyridine sulfonamideHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: SupramolecularOptimization: GeneticReaction: Transfer hydrogenationMax TON: 100ee: 96PDB: ---Notes: ---
Redox-Switchable Siderophore Anchor Enables Reversible Artificial Metalloenzyme Assembly
Nat. Catal. 2018, 1, 680-688, 10.1038/s41929-018-0124-3
Artificial metalloenzymes that contain protein-anchored synthetic catalysts are attracting increasing interest. An exciting, but still unrealized advantage of non-covalent anchoring is its potential for reversibility and thus component recycling. Here we present a siderophore–protein combination that enables strong but redox-reversible catalyst anchoring, as exemplified by an artificial transfer hydrogenase (ATHase). By linking the iron(iii)-binding siderophore azotochelin to an iridium-containing imine-reduction catalyst that produces racemic product in the absence of the protein CeuE, but a reproducible enantiomeric excess if protein bound, the assembly and reductively triggered disassembly of the ATHase was achieved. The crystal structure of the ATHase identified the residues involved in high-affinity binding and enantioselectivity. While in the presence of iron(iii), the azotochelin-based anchor binds CeuE with high affinity, and the reduction of the coordinated iron(iii) to iron(ii) triggers its dissociation from the protein. Thus, the assembly of the artificial enzyme can be controlled via the iron oxidation state.
Metal: IrLigand type: Cp*; Pyridine sulfonamideHost protein: CeuEAnchoring strategy: SupramolecularOptimization: Chemical & geneticReaction: Transfer hydrogenationMax TON: ---ee: 35.4PDB: 5OD5Notes: Redox switchable iron(III)-azotochelin anchor