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.

Creus, M.

Protein Expression Purif. 2011, 77, 131-139, 10.1016/j.pep.2011.01.003

The avidin–biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.

Creus, M.; Ward, T.R.

Prog. Inorg. Chem. 2011, 203-253, 10.1002/9781118148235.ch4

n/a

Alberto, R.; Ward, T.R.

Helv. Chim. Acta 2018, 101, e1800036, 10.1002/hlca.201800036

Photocatalytic hydrogen evolution by an artificial hydrogenase based on the biotin‐streptavidin technology is reported. A biotinylated cobalt pentapyridyl‐based hydrogen evolution catalyst (HEC) was incorporated into different mutants of streptavidin. Catalysis with [Ru(bpy)3]Cl2 as a photosensitizer (PS) and ascorbate as sacrificial electron donor (SED) at different pH values highlighted the impact of close lying amino acids that may act as a proton relay under the reaction conditions (Asp, Arg, Lys). In the presence of a close‐lying lysine residue, both, the rates were improved, and the reaction was initiated much faster. The X‐ray crystal structure of the artificial hydrogenase reveals a distance of 8.8 Å between the closest lying Co‐moieties. We thus suggest that the hydrogen evolution mechanism proceeds via a single Co centre. Our findings highlight that streptavidin is a versatile host protein for the assembly of artificial hydrogenases and their activity can be fine‐tuned via mutagenesis.