Marchi-Delapierre, C.

ACS Catal. 2020, 10, 5631-5645, 10.1021/acscatal.9b04904

Artificial enzymes represent an attractive alternative to design abiotic biocatalysis. EcNikA-Ru1, an artificial metalloenzyme developed by embedding a ruthenium-based catalyst into the cavity of the periplasmic nickel-binding protein NikA, was found to efficiently and selectively transform certain alkenes. The objective of this study was to provide a rationale on the enzymatic function and the unexpected substrate-dependent chemoselectivity of EcNikA-Ru1 thanks to a dual experimental/computational study. We observed that the de novo active site allows the formation of the terminal oxidant via the formation of a ruthenium aquo species that subsequently reacts with the hypervalent iodine of phenyl iodide diacetic acid. The oxidation process relies on a RuIV═O pathway via a two-step reaction with a radical intermediate, resulting in the formation of either a chlorohydrin or an epoxide. The results emphasize the impact of the protein scaffold on the kinetics of the reaction, through (i) the promotion of the starting oxidizing species via the exchange of a CO ligand with a water molecule; and (ii) the control of the substrate orientation on the intermediate structures, formed after the RuIV═O attack. When a Cα attack is preferred, chlorohydrins are formed while an attack on Cβ leads to an epoxide. This work provides evidence that artificial enzymes mimic the behavior of their natural counterparts.

Höcker, B.; Lechner, H.

ChemBioChem 2021, 22, 1573-1577, 10.1002/cbic.202000880

An artificial cofactor based on an organocatalyst embedded in a protein has been used to conduct the Baylis-Hillman reaction in a buffered system. As protein host, we chose streptavidin, as it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed on the basis of high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize the observed activity. Finally, repeated cycles of structure determination and redesign led to a system with an up to one order of magnitude increase in activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.

Shaw, W.J.

Organometallics 2020, 39, 1532-1544, 10.1021/acs.organomet.9b00843

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(PEt2NglycinePEt2)2]−, immobilized covalently within a protein scaffold was activated for CO2 hydrogenation. Here, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h–1, and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.